Lipids for delivery of therapeutics

ABSTRACT

This disclosure provides a range of amino acid lipid compounds and compositions useful for drug delivery, therapeutics, and the diagnosis and treatment of diseases and conditions. The amino acid lipid compounds and compositions can be used for delivery of various agents such as nucleic acid therapeutics to cells, tissues, organs, and subjects.

SEQUENCE LISTING

This application includes a Sequence Listing submitted herewith via EFSas an ASCII file created on Sep. 24, 2014, named MAR278US_Seq_List.txt,which is 7179 bytes in size, and is hereby incorporated by reference inits entirety.

BACKGROUND

The delivery of a therapeutic compound to a subject can be impeded bylimited ability of the compound to reach a target cell or tissue, or byrestricted entry or trafficking of the compound within cells. Deliveryof a therapeutic material is in general restricted by membranes ofcells. These barriers and restrictions to delivery can result in theneed to use much higher concentrations of a compound than is desirableto achieve a result, which brings the risk of toxic effects and sideeffects.

One strategy for delivery is to improve transport of a compound intocells using lipid or polymeric carrier molecules. These materials cantake advantage of mechanisms that exist for selective entry into a cell,while still excluding exogenous molecules such as nucleic acids andproteins. For example, a cationic lipid may interact with a drug agentand provide contact with a cell membrane. Lipid molecules can also beorganized into liposomes or particles as carriers for drug agents.Liposomal drug carriers can protect a drug molecule from degradationwhile improving its uptake by cells. Also, liposomal drug carriers canencapsulate or bind certain compounds by electrostatic and otherinteractions, and may interact with negatively charged cell membranes toinitiate transport across a membrane.

The understanding of regulatory RNA and the development of RNAinterference (RNAi), RNAi therapy, RNA drugs, antisense therapy, andgene therapy, among others, has increased the need for effective meansof introducing active nucleic acid agents into cells. In general,nucleic acids are stable for only limited times in cells or plasma.However, nucleic acid-based agents can be stabilized in compositions andformulations which may then be dispersed for cellular delivery.

This disclosure provides compounds, compositions, methods and uses forimproving systemic and local delivery of drugs and biologically activemolecules. Among other things, this application provides novel compoundsand compositions for making and using delivery structures and carrierswhich increase the efficiency of delivery of biologically activemolecules.

BRIEF SUMMARY

This disclosure provides novel compounds, compositions and formulationsfor intracellular and in vivo delivery of drug agents for use,ultimately, as a therapeutic. The compounds and compositions of thisdisclosure are useful for delivery of drug agents to selected cells,tissues, organs or compartments in order to alter a disease state or aphenotype.

In some aspects, this disclosure provides compounds, compositions andmethods to deliver RNA structures to cells to produce the response ofRNA interference, antisense effects, or the regulation of genomicexpression.

This invention provides a range of amino acid lipids which arelipophilic compounds for use in delivery and administration of drugagents and in drug delivery systems. The amino acid lipids of thisdisclosure are molecules containing an amino acid residue and one ormore lipophilic tails.

In some aspects, this invention provides a range of amino acid lipidcompounds as shown in Formula I:

R³—(C═O)-Xaa-Z—R⁴  Formula I

wherein

-   -   Xaa is any D- or L-amino acid residue having the formula        —NR^(N)—CR¹R²—(C═O)—, or a peptide of n=2-20 amino acid residues        having the formula —{NR^(N)—CR¹R²—(C═O)}_(n)—, wherein        -   R¹ is independently, for each occurrence, a non-hydrogen,            substituted or unsubstituted side chain of an amino acid;        -   R² is independently, for each occurrence, hydrogen, or an            organic group consisting of carbon, oxygen, nitrogen,            sulfur, and hydrogen atoms, and having from 1 to 20 carbon            atoms, or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl,            C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,            C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,            C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,            C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl,            cyano-C(1-5)alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl,            carboxyl, or hydroxyl,        -   R^(N) is independently, for each occurrence, hydrogen, or an            organic group consisting of carbon, oxygen, nitrogen,            sulfur, and hydrogen atoms, and having from 1 to 20 carbon            atoms, or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl,            C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,            C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,            C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,            C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl,            cyano-C(1-5)alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl,            carboxyl, or hydroxyl,    -   R³ is independently a lipophilic tail derived from a        naturally-occurring or synthetic lipid, phospholipid,        glycolipid, triacylglycerol, glycerophospholipid, sphingolipid,        ceramide, sphingomyelin, cerebroside, or ganglioside, wherein        the tail may contain a steroid; or a substituted or        unsubstituted C(3-22)alkyl, C(6-12)cycloalkyl,        C(6-12)cycloalkyl-C(3-22)alkyl, C(3-22)alkenyl, C(3-22)alkynyl,        C(3-22)alkoxy, or C(6-12)alkoxy-C(3-22)alkyl;    -   R⁴ is independently a lipophilic tail derived from a        naturally-occurring or synthetic lipid, phospholipid,        glycolipid, triacylglycerol, glycerophospholipid, sphingolipid,        ceramide, sphingomyelin, cerebroside, or ganglioside, wherein        the tail may contain a steroid; or a substituted or        unsubstituted C(3-22)alkyl, C(6-12)cycloalkyl,        C(6-12)cycloalkyl-C(3-22)alkyl, C(3-22)alkenyl, C(3-22)alkynyl,        C(3-22)alkoxy, or C(6-12)alkoxy-C(3-22)alkyl;    -   wherein either one of R³ and R⁴ is a lipophilic tail as defined        above and the other is an amino acid terminal group, or both R³        and R⁴ are lipophilic tails; the amino acid terminal group being        hydrogen, hydroxyl, amino, or an organic protective group;    -   Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker        consisting of 1-40 atoms selected from hydrogen, carbon, oxygen,        nitrogen, and sulfur atoms;        and salts thereof.

In some respects, this invention provides a range of amino acid lipidcompounds as shown in Formula I:

R³—(C═O)-Xaa-Z—R⁴  Formula I

wherein

-   -   Xaa is a D- or L-amino acid residue having the formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a substituted or unsubstituted basic side chain of an            amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is independently a substituted or unsubstituted C(6-22)alkyl        or C(6-22)alkenyl;    -   R⁴ is independently a substituted or unsubstituted C(6-22)alkyl        or C(6-22)alkenyl;    -   Z is NH, O, or an organic linker consisting of 1-40 atoms        selected from hydrogen, carbon, oxygen, nitrogen, and sulfur        atoms;        and salts thereof.

In some embodiments, the amino acid lipid compound may contain Xaaselected from arginine, homoarginine, norarginine, nor-norarginine,ornithine, lysine, homolysine, histidine, 1-methylhistidine,pyridylalanine, asparagine, N-ethylasparagine, glutamine,4-aminophenylalanine, the N-methylated versions thereof, and side chainmodified derivatives thereof. In some embodiments, the amino acid lipidcompound may contain Xaa selected from cysteine and serine.

In certain embodiments, R³ and R⁴ may be C(6-22)alkyl and may be thesame or different. In some embodiments, R³ and R⁴ may be C(6-22)alkenyland may be the same or different.

In certain embodiments, Xaa may be a peptide of 2-20 amino acidresidues.

In some embodiments, Xaa may have a side chain containing a functionalgroup having a pKa from 5 to 7.5.

In some aspects, the amino acid lipid compound may be a multi-mer of twoor more of the amino acid lipid compounds which are crosslinked.

In some embodiments, the amino acid lipid compound may be a conjugatehaving a peptide conjugated to the side chain of the amino acid residue.

In certain embodiments, the amino acid lipid compound may be attached toan oligomeric or polymeric framework.

In some embodiments, the amino acid lipid compound may be attached to apharmaceutical drug compound.

In some aspects, this invention provides a range of amino acid lipidcompounds as shown in Formula I:

R³—(C═O)-Xaa-Z—R⁴  Formula I

wherein

-   -   Xaa is a D- or L-amino acid residue having the formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a substituted or unsubstituted basic side chain of an            amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is independently a substituted or unsubstituted C(6-22)alkyl        or C(6-22)alkenyl;    -   R⁴ is hydrogen;    -   Z is NH, O, or an organic linker consisting of 1-40 atoms        selected from hydrogen, carbon, oxygen, nitrogen, and sulfur        atoms;        and salts thereof.

In some respects, this disclosure encompasses compositions containingone or more amino acid lipid compounds and one or more therapeuticnucleic acids. The therapeutic nucleic acid may be a gene silencingagent, or an RNAi-inducing agent, or a double-stranded RNA, or an mdRNA,or may contain a modified nucleoside.

In some embodiments, this disclosure encompasses compositions containingone or more amino acid lipid compounds and one or more additionalnon-amino acid lipids or polymeric lipids. In some embodiments, thecomposition may contain cholesteryl hemisuccinate.

In some aspects, this disclosure encompasses compositions containing oneor more amino acid lipid compounds and one or more nucleic acids whichmay form a complex with an amino acid lipid.

In certain embodiments, this disclosure encompasses compositionscontaining one or more amino acid lipid compounds which form liposomes.

In some aspects, this disclosure encompasses compositions containing oneor more amino acid lipid compounds which form an emulsion.

In some embodiments, this disclosure encompasses compositions containingone or more amino acid lipid compounds which form a micellar dispersion.

In certain aspects, this disclosure encompasses compositions containingone or more amino acid lipid compounds and one or more drug agents orbiologically active agents.

In some aspects, this disclosure encompasses methods for delivering atherapeutic nucleic acid to a cell by preparing a composition containingone or more amino acid lipid compounds and treating a cell with thecomposition.

In some embodiments, this disclosure encompasses methods for inhibitingexpression of a gene in a cell comprising preparing a compositioncontaining one or more amino acid lipid compounds and treating a cellwith the composition.

In certain aspects, this disclosure encompasses methods for inhibitingexpression of a gene in a mammal comprising preparing a compositioncontaining one or more amino acid lipid compounds and administering thecomposition to the mammal.

In some embodiments, this disclosure encompasses methods for treating adisease in a human, the disease being selected from rheumatoidarthritis, liver disease, encephalitis, bone fracture, heart disease,viral disease including hepatitis and influenza, and cancer, comprisingpreparing a composition containing one or more amino acid lipidcompounds and administering the composition to the human.

In some aspects, this disclosure encompasses uses of a compositioncontaining one or more amino acid lipid compounds in the preparation ofa medicament for treating a disease including rheumatoid arthritis,liver disease, encephalitis, bone fracture, heart disease, viral diseaseincluding hepatitis and influenza, and cancer.

In some embodiments, this disclosure encompasses uses of a compositioncontaining one or more amino acid lipid compounds for treating a diseaseselected from rheumatoid arthritis, liver disease, encephalitis, bonefracture, heart disease, viral disease including hepatitis andinfluenza, and cancer.

This summary, taken along with the detailed description of theinvention, as well as the figures, the appended examples and claims, asa whole, encompass the disclosure of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of a liposomal embodiment of thisinvention in which amino acid lipids form a bilayer vesicle 10 alongwith other lipids. In this embodiment, the outer layer of the liposomeis protected by polyethyleneglycol chains 20 attached to a head group ofone of the lipids. The outer layer of the liposome also presents aligand 30 for specific targeting of a cell or tissue. The liposomalvesicle contains, in this embodiment, a cargo of active interfering RNAcomponents including a condensed RNA nanoparticle 40, a two-stranded RNAduplex peptide conjugate 50, a three-stranded mdRNA 60, a dicersubstrate RNA 70, a dsRNA with a long overhang 80, and an siRNA withblunt ends 90, which are pooled in this embodiment.

FIG. 2: Transmission electron micrograph obtained on a JEOL 1230 TEM ofa liposomal embodiment of this invention showing spherical lipid bilayervesicle particles formed with the amino acid lipid C12-norArg-C12. Thelength marker of the micrograph is 0.5 micrometer, and the sample wasstained with 3% uranyl acetate. The lipid portion of the liposomalformulation was [C12-norArg(NH₃⁺Cl⁻)—C12/DSPC/cholesterol/DSPE-PEG-2000] in the amounts of[30%/20%/49%/1%] respectively, as a mole percent of total lipid. Theliposomes contained the antiinfluenza-active dicer substrate dsRNADX3030.

FIG. 3: In FIG. 3 is shown an example of PPIB gene knockdown activityobtained from an in vitro assay in A549 cells. The concentrationresponse at 25, 10, 1, and 0.1 nM RNA of the normalized PPIB mRNAexpression values for two amino acid lipid formulations of aninterfering-RNA were compared to results for RNAIMAX. Formulation 1 was[C12-norArg(NH₃Cl)—C12/DOPE/CHOL (50/32/18)] and Formulation 2 was[C12-norArg(NH₃Cl)—C12/CHEMS/DLPE (50/32/18)]. The PPIB gene knockdownby an interfering-RNA in an amino acid lipid composition of thisdisclosure can exceed that obtained with RNAIMAX.

FIG. 4: In FIG. 4 is shown an example of LacZ gene knockdown activityobtained from an in vitro assay in 9 L/LacZ cells. The concentrationresponse at 25, 10, 1, and 0.1 nM RNA of the normalizedbeta-galactosidase expression values for two amino acid lipidformulations of an interfering-RNA were compared to results for RNAIMAX.Formulation 1 was [C12-norArg(NH₃Cl)—C12/DOPE/CHOL (50/32/18)] andFormulation 2 was [C12-norArg(NH₃Cl)—C12/CHEMS/DLPE (50/32/18)]. TheLacZ gene knockdown by an interfering-RNA in an amino acid lipidcomposition of this disclosure can exceed that obtained with RNAIMAX.

FIG. 5: In FIG. 5 is shown an example of ApoB gene knockdown activityobtained from an in vitro assay in HepG2 cells. The concentrationresponse at 25, 2.5, and 0.25 nM RNA of the normalized ApoB mRNAexpression values for three amino acid lipid formulations of aninterfering-RNA are shown. Formulation 1 was [non-amino acid cationiclipid/DSPC/chol./DMPE-PEG2k (40/10/48/2)]. Formulation 2 and 3 were both[C18:1-norArg-C16/CHEMS/DLPE/DMPE-PEG2k (50/32/16/2)].

DETAILED DESCRIPTION

This disclosure relates generally to novel compounds, compositions anduses thereof for delivery of drug agents. The compounds and compositionsof this disclosure are useful for delivery of therapeutic agents toselected cells, tissues, organs or subjects.

This disclosure relates generally to the chemistry of lipids and uses oflipid-like structures and materials to effect drug delivery.

This invention relates to novel drug delivery enhancing agents includinglipids that are useful for delivering various molecules to cells. Thisinvention provides a range of compounds, compositions, formulations,methods and uses of such agents directed ultimately toward drugdelivery, therapeutics, and the diagnosis and treatment of diseases andconditions, including those that respond to modulation of geneexpression or activity in a subject. More specifically, this inventionrelates to compounds, liposomes, lamellar vesicles, emulsions, micelles,suspensions, particles, solutions and other forms of delivery enhancingcompositions and formulations, as well as therapeutic methods and usesfor these delivery materials.

The compounds and compositions of this disclosure are useful fordelivery of therapeutic, prophylactic, and diagnostic agents such asnucleic acids, polynucleotides, peptides, proteins, and small moleculecompounds and drugs.

The compounds and compositions of this disclosure are useful fordelivery of therapeutic agents in forms such as liposomes, lamellarvesicles, emulsions, micelles, suspensions, particles, and solutions.These forms may include nanoparticles of various diameters.

In some respects, the compounds and compositions of this disclosure areuseful for delivery of a therapeutic agent in a liposome. In theseembodiments the therapeutic agent may be referred to as the cargo. Forexample, FIG. 1 shows a schematic representation of a liposomalembodiment of this invention in which amino acid lipids form a bilayervesicle 10 along with other lipids. In this embodiment, the outer layerof the liposome is protected by polyethyleneglycol chains 20 attached toa head group of one of the lipids. The outer layer of the liposome alsopresents a ligand 30 for specific targeting of a cell or tissue. Theliposomal vesicle contains, in this embodiment, a cargo of activeinterfering RNA components including a condensed RNA nanoparticle 40, atwo-stranded RNA duplex peptide conjugate 50, a three-stranded mdRNA 60,a dicer substrate RNA 70, a dsRNA with a long overhang 80, and an siRNAwith blunt ends 90, which are pooled in this embodiment. Other forms oftherapeutic cargo may include microRNA or hairpin RNA forms.

In some aspects, compounds and compositions of this disclosure mayprovide delivery of therapeutic agents in releasable forms orcompositions. Releasable forms and compositions include molecules thatbind and release an active agent, molecules that bind an active agentand discharge a moiety that assists in release of the agent, moleculesthat bind an active agent and are subsequently modulated in form withina biological compartment to assist in release of the agent, andcompositions containing molecules that bind an active agent admixed witha release mediator compound.

Amino Acid Lipids

This invention provides a range of amino acid lipids which arelipophilic compounds for use in delivery and administration of drugagents and in drug delivery systems. The amino acid lipids of thisdisclosure are molecules containing an amino acid residue and one ormore lipophilic tails.

In some embodiments, amino acid lipids are molecules having ahydrophilic portion and a lipophilic portion. The hydrophilic portionmay be provided by an amino acid residue. The lipophilic portion cancontain one or more lipophilic tails.

In some embodiments, amino acid lipids are lipophilic moleculescontaining a hydrophobic amino acid residue and one or more lipophilictails.

In some embodiments, the amino acid lipids provide relatively lowcytotoxicity, and correspondingly, a cytoprotective effect relative tocertain other lipids. In some embodiments, the amino acid lipids arepharmaceutically-acceptable, biodegradable, or biocompatable.

Amino acid lipids may be formed by substituting a delivery-enhancing orlipid-like tail at either the N-terminus or the C-terminus of an aminoacid, or at both termini. In some embodiments, the amino acid core mayinclude one or more amino acids, or may be a peptide of 2-20 amino acidresidues.

Amino acid lipids can be cationic or non-cationic, where non-cationicincludes neutral and anionic. As used herein, the physical state orionicity of a species refers to an environment having pH about 7, unlessotherwise specified.

Amino acid lipids of this disclosure may exhibit low cytotoxicity. Insome embodiments, amino acid lipids of this disclosure may providecytoprotective effects relative to lipids of other structures.

In some aspects, amino acid lipids of this disclosure may providedelivery of a therapeutic agent in a releasable form. Releasable formsand compositions are designed to provide sufficient uptake of an agentby a cell to provide a therapeutic effect.

Releasable forms include amino acid lipids that bind and release anactive agent. In some embodiments, release of the active agent may beprovided by an acid-labile linker.

Examples of acid-labile linkers include linkers containing an orthoestergroup, a hydrazone, a cis-acetonyl, an acetal, a ketal, a silyl ether, asilazane, an imine, a citriconic anhydride, a maleic anhydride, a crownether, an azacrown ether, a thiacrown ether, a dithiobenzyl group, acis-aconitic acid, a cis-carboxylic alkatriene, methacrylic acid, andmixtures thereof.

Examples of acid-labile groups and linkers are given in U.S. Pat. Nos.7,098,032; 6,897,196; 6,426,086; 7,138,382; 5,563,250; and 5,505,931.

Releasable forms of compounds and compositions of this disclosureinclude molecules that bind an active agent and discharge a moiety thatassists in release of the agent. In some embodiments, an amino acidlipid may include a group which releases a small molecule such asethanol that assists in delivering an agent to a cell. An amino acidlipid may bind an active agent and, subsequent to contact with a cell,or subsequent to transport within a biological compartment having alocal pH lower than physiological pH, be hydrolyzed in an acidicenvironment to release ethanol to assist in delivery of the agent. Insome embodiments, a small molecule such as ethanol, which assists indelivery of the agent, may be bound to a lipid component.

In some embodiments, an amino acid lipid may be admixed with a compoundthat releases a small molecule such as ethanol to assists in deliveringan agent to a cell.

Releasable forms of compounds and compositions of this disclosureinclude amino acid lipids which may bind an active agent and, subsequentto contact with a cell, or subsequent to transport within a biologicalcompartment having a local pH lower than physiological pH, be modulatedin an acidic environment into a cationic form to assist in release ofthe agent.

In some embodiments, an amino acid lipid may bind an active agent, andmay be admixed with a compound that can be modulated in an acidicenvironment into a cationic form to assist in release of an activeagent.

Examples of hydrolysable and modulatable groups are given in U.S. Pat.Nos. 6,849,272; 6,200,599; as well as Z. H. Huang and F. C. Szoka,“Bioresponsive liposomes and their use for macromolecular delivery,” in:G. Gregoriadis (ed.), Liposome Technology, 3rd ed. (CRC Press 2006).

In some embodiments, releasable forms of compounds and compositions ofthis disclosure include amino acid lipids which can bind an activeagent, and may be admixed with a lipid or compound that can be modulatedin an acidic environment into a neutral form to assist in release of anactive agent. The acidic environment may be entered subsequent tocontact with a cell, or subsequent to transport within a biologicalcompartment having a local pH lower than physiological pH.

Examples of lipids which are modulatable from anionic to neutral formsinclude cholesteryl hemisuccinate (CHEMS) as described in U.S. Pat. Nos.6,897,196; 6,426,086; and 7,108,863.

In some embodiments, releasable forms of compounds and compositions ofthis disclosure include amino acid lipids which can bind an activeagent, and may be admixed with a pH-sensitive polymeric material.

Examples of pH-sensitive polymeric materials are given in U.S. Pat. No.6,835,393.

In some embodiments, release of the active agent may be provided by anenzyme-cleavable peptide.

In some aspects, this invention provides a range of amino acid lipids asshown in Formula I:

R³—(C═O)-Xaa-Z—R⁴  Formula I

wherein

-   -   Xaa is any D- or L-amino acid residue having the formula        —NR^(N)—CR¹R²—(C═O)—, or a peptide of n=2-20 amino acid residues        having the formula —{NR^(N)—CR¹R²—(C═O)}_(n)—, wherein        -   R¹ is independently, for each occurrence, a non-hydrogen,            substituted or unsubstituted side chain of an amino acid;        -   R² is independently, for each occurrence, hydrogen, or an            organic group consisting of carbon, oxygen, nitrogen,            sulfur, and hydrogen atoms, and having from 1 to 20 carbon            atoms, or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl,            C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,            C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,            C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,            C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl,            cyano-C(1-5)alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl,            carboxyl, or hydroxyl,        -   R^(N) is independently, for each occurrence, hydrogen, or an            organic group consisting of carbon, oxygen, nitrogen,            sulfur, and hydrogen atoms, and having from 1 to 20 carbon            atoms, or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl,            C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,            C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,            C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,            C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl,            cyano-C(1-5)alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl,            carboxyl, or hydroxyl,    -   R³ is a lipophilic tail derived from a naturally-occurring or        synthetic phospholipid, glycolipid, triacylglycerol,        glycerophospholipid, sphingolipid, ceramide, sphingomyelin,        cerebroside, or ganglioside; or a substituted or unsubstituted        C(3-22)alkyl, C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl; or a lipophilic tail of any other        naturally-occurring or synthetic lipid, or a lipophilic tail of        any one of the additional delivery lipids described hereinbelow,        and may contain a steroid;    -   R⁴ is a lipophilic tail derived from a naturally-occurring or        synthetic phospholipid, glycolipid, triacylglycerol,        glycerophospholipid, sphingolipid, ceramide, sphingomyelin,        cerebroside, or ganglioside; or substituted or unsubstituted        C(3-22)alkyl, C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl; or a lipophilic tail of any other        naturally-occurring or synthetic lipid, or a lipophilic tail of        any one of the additional delivery lipids described hereinbelow,        and may contain a steroid;    -   Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker        consisting of 1-40 atoms selected from hydrogen, carbon, oxygen,        nitrogen, and sulfur atoms;        and salts thereof.

In some embodiments, R³ is independently a substituted or unsubstitutedC(6-22)alkyl or C(6-22)alkenyl; R⁴ is independently a substituted orunsubstituted C(6-22)alkyl or C(6-22)alkenyl.

The residue Xaa may be a D- or L-stereocenter.

In some embodiments, the amino acid core may be a peptide of 2-20 aminoacid residues having lipophilic tails at the N-terminus and C-terminusof the peptide.

In some embodiments, R¹ is a non-hydrogen, substituted or unsubstitutedside chain of an amino acid wherein a substituent of a side chain is anorganic group consisting of 1 to 40 atoms selected from hydrogen,carbon, oxygen, nitrogen, and sulfur atoms.

In some embodiments, Z is an alkyl or an organic linker syntheticpolymer such as a polyethylene glycol chain (PEG), or a PEG copolymersuch as PEG-polyurethane or PEG-polypropylene. See, e.g., J. MiltonHarris, Poly(ethylene glycol) chemistry: biotechnical and biomedicalapplications (1992).

In some embodiments, this invention provides a range of amino acidlipids as shown in Formula I above wherein:

-   -   Xaa is any D- or L-amino acid having the general formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a non-hydrogen, substituted or unsubstituted basic            side chain of an amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is a lipophilic tail derived from a naturally-occurring or        synthetic phospholipid, glycolipid, triacylglycerol,        glycerophospholipid, sphingolipid, ceramide, sphingomyelin,        cerebroside, or ganglioside; or a substituted or unsubstituted        C(3-22)alkyl, C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl; or a lipophilic tail of any other        naturally-occurring or synthetic lipid, or a lipophilic tail of        any one of the additional delivery lipids described hereinbelow,        and may contain a steroid;    -   R⁴ is a lipophilic tail derived from a naturally-occurring or        synthetic phospholipid, glycolipid, triacylglycerol,        glycerophospholipid, sphingolipid, ceramide, sphingomyelin,        cerebroside, or ganglioside; or substituted or unsubstituted        C(3-22)alkyl, C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl; or a lipophilic tail of any other        naturally-occurring or synthetic lipid, or a lipophilic tail of        any one of the additional delivery lipids described hereinbelow,        and may contain a steroid;    -   Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker        consisting of 1-40 atoms selected from hydrogen, carbon, oxygen,        nitrogen, and sulfur atoms.

In some embodiments, this invention provides a range of amino acidlipids as shown in Formula I above wherein:

-   -   Xaa is any D- or L-amino acid having the general formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a non-hydrogen, substituted or unsubstituted basic            side chain of an amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   R⁴ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker        consisting of 1-40 atoms selected from hydrogen, carbon, oxygen,        nitrogen, and sulfur atoms.

In some embodiments, this invention provides a range of amino acidlipids as shown in Formula I above wherein:

-   -   Xaa is any D- or L-amino acid having the general formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a non-hydrogen, substituted or unsubstituted basic            side chain of an amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   R⁴ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   Z is NH.

In some embodiments, this invention provides a range of amino acidlipids as shown in Formula I above wherein:

-   -   Xaa is any D- or L-amino acid having the general formula        —NR^(N)—CR¹R²—(C═O)—, wherein        -   R¹ is a non-hydrogen, substituted or unsubstituted basic            side chain of an amino acid;        -   R² is hydrogen, or C(1-5)alkyl,        -   R^(N) is hydrogen, or C(1-5)alkyl,    -   R³ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   R⁴ is a substituted or unsubstituted C(3-22)alkyl,        C(6-12)cycloalkyl, C(6-12)cycloalkyl-C(3-22)alkyl,        C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or        C(6-12)alkoxy-C(3-22)alkyl;    -   Z is O.

Cationic amino acid lipids can be prepared where, for example, Xaa has abasic side chain. Examples of amino acids having a basic side chaininclude arginine (Arg), homoarginine (homoArg) (side chain—(CH₂)₄NH(C═NH)NH₂), norarginine (norArg) (side chain—(CH₂)₂NH(C═NH)NH₂), nor-norarginine (nornorArg) (side chain—(CH₂)NH(C═NH)NH₂), ornithine, lysine, homolysine, histidine,1-methylhistidine, pyridylalanine (Pal), asparagine, N-ethylasparagine,glutamine, and 4-aminophenylalanine, and side chain modified derivativesthereof.

As used herein, the term “homo,” when referring to an amino acid, meansthat an additional carbon is added to the side chain, while the term“nor,” when referring to an amino acid, means that a carbon issubtracted from the side chain. Thus, homolysine refers to sidechain-(CH₂)₅NH₂.

Anionic amino acid lipids can be prepared where, for example, Xaa isglutamate or aspartate.

Cationic and anionic amino acid lipids can also be prepared where theamino acid side chain contains an ionizable group or substituent.

Non-cationic amino acid lipids can be prepared where, for example, Xaais leucine, valine, alanine, or serine.

In some embodiments, Xaa is N^(G)-methylarginine, symmetric orasymmetric N^(G),N^(G)-dimethylarginine, N^(G)-methyl-homoarginine,symmetric or asymmetric N^(G),N^(G)-dimethyl-homoarginine,N^(G)-methyl-norarginine, symmetric or asymmetricN^(G),N^(G)-dimethyl-norarginine, or N^(G)-methyl-nor-norarginine,symmetric or asymmetric N^(G),N^(G)-dimethyl-nor-norarginine.

In some embodiments, Xaa is N^(G)-ethylarginine, symmetric or asymmetricN^(G),N^(G)-diethylarginine, N^(G)-ethyl-homoarginine, symmetric orasymmetric N^(G),N^(G)-diethyl-homoarginine, N^(G)-ethyl-norarginine,symmetric or asymmetric N^(G),N^(G)-diethyl-norarginine, orN^(G)-ethyl-nor-norarginine, symmetric or asymmetricN^(G),N^(G)-diethyl-nor-norarginine.

In certain embodiments, Xaa is N^(G)-alkylarginine, symmetric orasymmetric N^(G),N^(G)-dialkylarginine, N^(G)-alkyl-homoarginine,symmetric or asymmetric N^(G),N^(G)-dialkyl-homoarginine,N^(G)-alkyl-norarginine, symmetric or asymmetricN^(G),N^(G)-dialkyl-norarginine, or N^(G)-alkyl-nor-norarginine,symmetric or asymmetric N^(G),N^(G)-dialkyl-nor-norarginine.

In some embodiments, Xaa is an amino acid having a guanidine- oramidine-containing side chain. For example, the side chain of the Xaaresidue may contain a group such as guanido, amidino, dihydroimidazole,4-guanido-phenyl, 4-amidino-phenyl, N-amidino-piperidine,N-amidino-piperazine, 4,5-dihydroimidazole, 2-(N-amidino)-pyrrolidinyl,or 4-[(2-aminopyrimidinyl)]ethyl.

Examples of Xaa side chains include the following structures, as well astheir salt forms:

Examples of a substituted side chain of an amino acid suitable for areleasable form of an amino acid lipid include a releasing functionalgroup having a pKa from about 5 to about 7.5, or from about 6 to about7. In general, a releasing functional group which is a weak base mayexhibit a predominant neutral form at a local pH above pKa, and mayexhibit a predominant ionic form at a local pH below pKa. A releasingfunctional group which is a weak acid may exhibit an ionic form at alocal pH above pKa, and may exhibit a neutral form at a local pH belowpKa. See, e.g., P. Heinrich Stahl, Handbook of Pharmaceutical Salts,(2002).

In some embodiments, Xaa may have a side chain containing a functionalgroup having a pKa from 5 to 7.5.

Examples of a substituted side chain of an amino acid suitable for areleasable form of an amino acid lipid include 1-methylhistidine.

Examples of a substituted side chain of an amino acid suitable for areleasable form of an amino acid lipid include 3,5-diiodo-tyrosine.

Examples of a substituted side chain of an amino acid suitable for areleasable form of an amino acid lipid include the following structures:

Examples of amino acid lipids include the structures:

Examples of a substituent on a side chain of an amino acid suitable fora releasable form of an amino acid lipid include releasing functionalgroups derived from 3,5-diiodo-tyrosine, 1-methylhistidine,2-methylbutanoic acid, 2-o-anisylpropanoic acid, meso-tartaric acid,4,6-dimethylpyrimidinamine, p-phthalic acid, creatinine, butanoic acid,N,N-dimethyl-1-naphthylamine, pentanoic acid, 4-methylpentanoic acid,N-methylaniline, 1,10-phenanthroline, 3-pyridinecarboxylic acid,hexanoic acid, propanoic acid, 4-animobenzoic acid, 2-methylpropanoicacid, heptanoic acid, octanoic acid, cyclohexanecarboxylic acid,quinoline, 3-quinolinamine, 2-aminobenzoic acid, 4-pyridinecarboxylicacid, nonanic acid, melamine, 8-quinolinol, trimethylacetic acid,6-methoxyquinoline, 4-(methylamino)benzoic acid, p-methylaniline,3-(methylamino)benzoic acid, malic acid, N-ethylaniline,2-benzylpyridine, 3,6-dinitrophenol, N,N-dimethylaniline,2,5-dimethylpiperazine, p-phenetidine, 5-methylquinoline,2-phenylbenzimidazole, pyridine, picolinic acid, 3,5-diiodityrosine,p-anisidine, 2-(methylamino)benzoic acid, 2-thiazolamine, glutaric acid,adipic acid, isoquinoline, itaconic acid, o-phthalic acid,benzimidazole, piperazine, heptanedioic acid, acridine, phenanthridine,succinic acid, methylsuccinic acid, 4-methylquinoline, 3-methylpyridine,7-isoquinolinol, malonic acid, methymalonic acid, 2-methylquinoline,2-ethylpyridine, 2-methylpyridine, 4-methylpyridine, histamine,histidine, maleic acid, cis-1,2-cyclohexanediamine,3,5-dimethylpyridine, 2-ethylbenzimidazole, 2-methylbenzimidazole,cacodylic acid, perimidine, citric acid, isocitric acid,2,5-dimethylpyridine, papaverine, 6-hydroxy-4-methylpteridine,L-thyroxine, 3,4-dimethylpyridine, methoxypyridine,trans-1,2-cyclohexanediamine, 2,5-pyridinediamine, l-1-methylhistidine,l-3-methylhistidine, 2,3-dimethylpyridine, xanthopterin,1,2-propanediamine, N,N-diethylaniline, alloxanic acid,2,6-dimethylpyridine, L-carnosine, 2-pyridinamine, N-b-alanylhistidine,pilocarpine, 1-methylimidazol, 1H-imidazole, 2,4-dimethylpyridine,4-nitrophenol, 2-nitrophenol, tyrosineamide, 5-hydoxxyquinazoline,1,1-cyclopropanedicarboxylic acid, 2,4,6-trimethylpyridine, veronal,2,3-dichlorophenol, 1,2-ethanediamine, 1-isoquinolinamine, andcombinations thereof.

In some embodiments, a range of amino acid lipids corresponding toFormula I are represented by the structures

where R¹, R², R^(N), R³, and R⁴ are defined as above.

In some embodiments, R³ and R⁴ are independently selected lipid-liketails which impart sufficient lipophilic character or lipophilicity,such as defined by water/octanol partitioning, to provide deliveryacross a membrane or uptake by a cell. These tails provide, when used inan amino acid lipid structure, an amphipathic molecule. Lipid-like tailsmay be derived from phospholipids, glycolipids, triacylglycerols,glycerophospholipids, sphingolipids, ceramides, sphingomyelins,cerebrosides, or gangliosides, among others, and may contain a steroid.

In certain embodiments, R³ and R⁴ may independently be a lipid-like tailhaving a glycerol backbone.

In some embodiments, R³ and R⁴ may independently be C3alkyl, C4alkyl,C5alkyl, C6alkyl, C7alkyl, C8alkyl, C9alkyl, C10alkyl, C11alkyl,C12alkyl, C13alkyl, C14alkyl, C15alkyl, C16alkyl, C17alkyl, C18alkyl,C19alkyl, C20alkyl, C21alkyl, or C22alkyl.

In some embodiments, R³ and R⁴ may independently be lipophilic tailshaving one of the following structures:

In the structures above, X represents the atom of the tail that isdirectly attached to the amino acid residue terminus, and is counted asone of the atoms in the numerical designation, for example, “18:3.” Insome embodiments, X may be a carbon, nitrogen, or oxygen atom.

In some embodiments, R³ and R⁴ may independently be lipophilic tailshaving one of the following structures:

where X is as defined above.

In some embodiments, R³ and R⁴ are independently selected lipid-liketails which may contain a cholesterol, a sterol, or a steroid such asgonanes, estranes, androstanes, pregnanes, cholanes, cholestanes,ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes,lanostanes, cycloartanes, as well as sterol or zoosterol derivatives ofany of the foregoing, and their biological intermediates and precursors,which may include, for example, cholesterol, lanosterol, stigmastanol,dihydrolanosterol, zymosterol, zymostenol, desmosterol,7-dehydrocholesterol, and mixtures and derivatives thereof.

In certain embodiments, R³ and R⁴ may independently be derived fromfatty acid-like tails such as tails from myristic acid (C14:0)alkenyl,palmitic acid (C16:0)alkenyl, stearic acid (C18:0)alkenyl, oleic acid(C18:1, double bond at carbon 9)alkenyl, linoleic acid (C18:2, doublebond at carbon 9 or 12)alkenyl, linonenic acid (C18:3, double bond atcarbon 9, 12, or 15)alkenyl, arachidonic acid (C20:4, double bond atcarbon 5, 8, 11, or 14)alkenyl, and eicosapentaenoic acid (C20:5, doublebond at carbon 5, 8, 11, 14, or 17)alkenyl. Other examples of fattyacid-like tails are found at Donald Voet and Judith Voet, Biochemistry,3rd Edition (2005), p. 383.

In some embodiments, R³ and R⁴ may independently be derived from anisoprenoid.

As used herein, the term “amino acid” includes naturally-occurring andnon-naturally occurring amino acids. Thus, an amino acid lipid of thisinvention can be made from a genetically encoded amino acid, a naturallyoccurring non-genetically encoded amino acid, or a synthetic amino acid.

Examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

Examples of amino acids include azetidine, 2-aminooctadecanoic acid,2-aminoadipic acid, 3-aminoadipic acid, 2,3-diaminopropionic acid,2-aminobutyric acid, 4-aminobutyric acid, 2,3-diaminobutyric acid,2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid,2-aminopimelic acid, 2,2′-diaminopimelic acid, 6-aminohexanoic acid,6-aminocaproic acid, 2-aminoheptanoic acid, desmosine, ornithine,citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine,t-butylglycine, N-methylglycine, sacrosine, N-ethylglycine,cyclohexylglycine, 4-oxo-cyclohexylglycine, N-ethylasparagine,cyclohexylalanine, t-butylalanine, naphthylalanine, pyridylalanine,3-chloroalanine, 3-benzothienylalanine, 4-halophenylalanine,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine,4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine,methionine sulfoxide, homoarginine, norarginine, nor-norarginine,N-acetyllysine, 4-aminophenylalanine, N-methylvaline, homocysteine,homoserine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine,norvaline, O-allyl-serine, O-allyl-threonine, alpha-aminohexanoic acid,alpha-aminovaleric acid, pyroglutamic acid, and derivatives thereof.

As used herein, the term “amino acid” includes alpha- and beta-aminoacids.

Examples of amino acid residues can be found in Fasman, CRC PracticalHandbook of Biochemistry and Molecular Biology, CRC Press, Inc. (1989).

In general, a compound may contain one or more chiral centers. Compoundscontaining one or more chiral centers may include those described as an“isomer,” a “stereoisomer,” a “diastereomer,” an “enantiomer,” an“optical isomer,” or as a “racemic mixture.” Conventions forstereochemical nomenclature, for example the stereoisomer naming rulesof Cahn, Ingold and Prelog, as well as methods for the determination ofstereochemistry and the separation of stereoisomers are known in theart. See, for example, Michael B. Smith and Jerry March, March'sAdvanced Organic Chemistry, 5th edition, 2001. The compounds andstructures of this disclosure are meant to encompass all possibleisomers, stereoisomers, diastereomers, enantiomers, and/or opticalisomers that would be understood to exist for the specified compound orstructure, including any mixture, racemic or otherwise, thereof.

Examples of amino acid lipids include R³—(C═O)-Arg-NH—R⁴ wherein Arg isD- or L-arginine, and R³ and R⁴ are independently alkyl or alkenyl.

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-norArg-NH—R⁴ whereinnorArg is D- or L-norarginine, and R³ and R⁴ are independently alkyl oralkenyl.

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-nornorArg-NH—R⁴ whereinnornorArg is D- or L-nor-norarginine, and R³ and R⁴ are independentlyalkyl such as heptyl, octyl, nonyl, decyl, and undecyl.

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-homoArg-NH—R⁴ whereinhomoArg is D- or L-homoarginine, and R³ and R⁴ are independently alkylsuch as heptyl, octyl, nonyl, decyl, and undecyl.

Examples of amino acid lipids include R³—(C═O)-4-pyridylalanine-NH—R⁴wherein the pyridylalanine is D- or L-pyridylalanine, and R³ and R⁴ areindependently alkyl such as heptyl, octyl, nonyl, decyl, and undecyl.Examples of R³—(C═O)-pyridylalanine-NH—R⁴ amino acid lipids includepharmaceutically-acceptable pyridyl salts, such as4-[N-methylpyridyl]alanine chloride. Examples of pyridylalanine aminoacid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-Lys-NH—R⁴ wherein R³ andR⁴ are independently alkyl or alkenyl.

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-His-NH—R⁴ wherein R³ andR⁴ are independently alkyl or alkenyl. Examples of His amino acid lipidsinclude the following structures:

Examples of amino acid lipids include R³—(C═O)-Xaa-O—R⁴ wherein R³ isalkyl and R⁴ is a sphingoid.

Examples of amino acid lipids include the following structures:

Examples of amino acid lipids include R³—(C═O)-Xaa-NH—R⁴ wherein R³ andR⁴ are alkyl or alkenyl. Examples of amino acid lipids include thefollowing structure:

Examples of amino acid lipids include (C10acyl)-Arg-NH—(C10alkyl),(C12acyl)-Arg-NH—(C12alkyl), (C14acyl)-Arg-NH—(C14alkyl),(C16acyl)-Arg-NH—(C16alkyl), (C18acyl)-Arg-NH—(C18alkyl),(C10acyl)-homoArg-NH—(C10alkyl), (C12acyl)-homoArg-NH—(C12alkyl),(C14acyl)-homoArg-NH—(C14alkyl), (C16acyl)-homoArg-NH—(C16alkyl),(C18acyl)-homoArg-NH—(C18alkyl), (C10acyl)-norArg-NH—(C10alkyl),(C12acyl)-norArg-NH—(C12alkyl), (C14acyl)-norArg-NH—(C14alkyl),(C16acyl)-norArg-NH—(C16alkyl), (C18acyl)-norArg-NH—(C18alkyl),(C10acyl)-nomorArg-NH—(C10alkyl), (C12acyl)-nornorArg-NH—(C12alkyl),(C14acyl)-nomorArg-NH—(C14alkyl), (C16acyl)-nornorArg-NH—(C16alkyl),(C18acyl)-nomorArg-NH—(C18alkyl), (C10acyl)-4-Pal-NH—(C10alkyl),(C12acyl)-4-Pal-NH—(C12alkyl), (C14acyl)-4-Pal-NH—(C14alkyl),(C16acyl)-4-Pal-NH—(C16alkyl), (C18acyl)-4-Pal-NH—(C18alkyl),(C10acyl)-4-Pal(Me)-NH—(C10alkyl), (C12acyl)-4-Pal(Me)-NH—(C12alkyl),(C14acyl)-4-Pal(Me)-NH—(C14alkyl), (C16acyl)-4-Pal(Me)-NH—(C16alkyl),and (C18acyl)-4-Pal(Me)-NH—(C18alkyl).

In general, the designation “C14-norArg-C14,” for example, refers to(C13alkyl)-(C═O)-norArg-NH—(C14alkyl) which is the same as(C14acyl)-norArg-NH—(C14alkyl).

Examples of amino acid lipids include(C10acyl)-D-Arg-L-Arg-NH—(C10alkyl),(C12acyl)-D-Arg-L-Arg-NH—(C12alkyl),(C14acyl)-D-Arg-L-Arg-NH—(C14alkyl),(C16acyl)-D-Arg-L-Arg-NH—(C16alkyl),(C18acyl)-D-Arg-L-Arg-NH—(C18alkyl),(C10acyl)-D-homoArg-L-homoArg-NH—(C10alkyl),(C12acyl)-D-homoArg-L-homoArg-NH—(C12alkyl),(C14acyl)-D-homoArg-L-homoArg-NH—(C14alkyl),(C16acyl)-D-homoArg-L-homoArg-NH—(C16alkyl),(C18acyl)-D-homoArg-L-homoArg-NH—(C18alkyl),(C10acyl)-D-norArg-L-norArg-NH—(C10alkyl),(C12acyl)-D-norArg-L-norArg-NH—(C12alkyl),(C14acyl)-D-norArg-L-norArg-NH—(C14alkyl),(C16acyl)-D-norArg-L-norArg-NH—(C16alkyl),(C18acyl)-D-norArg-L-norArg-NH—(C18alkyl),(C10acyl)-D-nornorArg-L-nornorArg-NH—(C10alkyl),(C12acyl)-D-nomorArg-L-nornorArg-NH—(C12alkyl),(C14acyl)-D-nomorArg-L-nornorArg-NH—(C14alkyl),(C16acyl)-D-nornorArg-L-nornorArg-NH—(C16alkyl),(C18acyl)-D-nornorArg-L-nornorArg-NH—(C18alkyl).

Examples of amino acid lipids include (C10acyl)-His-Arg-NH—(C10alkyl),(C12acyl)-His-Arg-NH—(C12alkyl), (C14acyl)-His-Arg-NH—(C14alkyl),(C16acyl)-His-Arg-NH—(C16alkyl), (C18acyl)-His-Arg-NH—(C18alkyl),(C10acyl)-His-Arg-NH—(C10alkyl), (C12acyl)-His-Arg-NH—(C12alkyl),(C14acyl)-His-Arg-NH—(C14alkyl), (C16acyl)-His-Arg-NH—(C16alkyl),(C18acyl)-His-Arg-NH—(C18alkyl), (C10acyl)-His-Arg-(C10alkyl),(C12acyl)-His-Arg-NH—(C12alkyl), (C14acyl)-His-Arg-NH—(C14alkyl),(C16acyl)-His-Arg-NH—(C16alkyl), (C18acyl)-His-Arg-NH—(C18alkyl),(C10acyl)-His-Arg-NH—(C10alkyl), (C12acyl)-His-Arg-NH—(C12alkyl),(C14acyl)-His-Arg-NH—(C14alkyl), (C16acyl)-His-Arg-NH—(C16alkyl),(C18acyl)-His-Arg-NH—(C18alkyl).

Examples of amino acid lipids include (C10acyl)-His-Asp-NH—(C10alkyl),(C12acyl)-His-Asp-NH—(C12alkyl), (C14acyl)-His-Asp-NH—(C14alkyl),(C16acyl)-His-Asp-NH—(C16alkyl), (C18acyl)-His-Asp-NH—(C18alkyl),(C10acyl)-His-Asp-NH—(C10alkyl), (C12acyl)-His-Asp-NH—(C12alkyl),(C14acyl)-His-Asp-NH—(C14alkyl), (C16acyl)-His-Asp-NH—(C16alkyl),(C18acyl)-His-Asp-NH—(C18alkyl), (C10acyl)-His-Asp-(C10alkyl),(C12acyl)-His-Asp-NH—(C12alkyl), (C14acyl)-His-Asp-NH—(C14alkyl),(C16acyl)-His-Asp-NH—(C16alkyl), (C18acyl)-His-Asp-NH—(C18alkyl),(C10acyl)-His-Asp-NH—(C10alkyl), (C12acyl)-His-Asp-NH—(C12alkyl),(C14acyl)-His-Asp-NH—(C14alkyl), (C16acyl)-His-Asp-NH—(C16alkyl),(C18acyl)-His-Asp-NH—(C18alkyl).

Examples of amino acid lipids include (C10acyl)-Pal-Arg-NH—(C10alkyl),(C12acyl)-Pal-Arg-NH—(C12alkyl), (C14acyl)-Pal-Arg-NH—(C14alkyl),(C16acyl)-Pal-Arg-NH—(C16alkyl), (C18acyl)-Pal-Arg-NH—(C18alkyl),(C10acyl)-Pal-Arg-NH—(C10alkyl), (C12acyl)-Pal-Arg-NH—(C12alkyl),(C14acyl)-Pal-Arg-NH—(C14alkyl), (C16acyl)-Pal-Arg-NH—(C16alkyl),(C18acyl)-Pal-Arg-NH—(C18alkyl), (C10acyl)-Pal-Arg-(C10alkyl),(C12acyl)-Pal-Arg-NH—(C12alkyl), (C14acyl)-Pal-Arg-NH—(C14alkyl),(C16acyl)-Pal-Arg-NH—(C16alkyl), (C18acyl)-Pal-Arg-NH—(C18alkyl),(C10acyl)-Pal-Arg-NH—(C10alkyl), (C12acyl)-Pal-Arg-NH—(C12alkyl),(C14acyl)-Pal-Arg-NH—(C14alkyl), (C16acyl)-Pal-Arg-NH—(C16alkyl),(C18acyl)-Pal-Arg-NH—(C18alkyl).

Amino acid lipids can be prepared as poly-mer or multi-mer species, suchas dimers, trimers, or tetramers. The poly-mer or multi-mer species canbe prepared from a single amino acid lipid, or from more than onespecies. Poly-mer or multi-mer amino acid lipids species can be preparedin some embodiments by providing a sulfhydryl group or othercross-linkable group on a side chain of the amino acid, or with linkedor tethered amino acid structures such as desmosine or citrulline. Inother embodiments, a poly-mer or multi-mer amino acid lipid species canbe prepared with bioconjugate linker chemistries.

Examples of amino acid lipids include the following structures:

An amino acid lipid can be prepared as a conjugate having a peptide orpolymer chain covalently attached to the amino acid side chain. Thepeptide or polymer chain can be attached using a reactive group of theamino acid side chain, for example, using the thiol or methylmercaptangroup of cysteine or methionine, respectively, or the alcohol group ofserine, or the amino group of lysine. The peptide or polymer chain canbe attached using any reactive group of a substituted or modified aminoacid side chain. Various linker groups such as NHS, maleimido, andbioconjugate techniques and linkers can be used.

Amino acid lipids can be prepared as constructs attached to anoligomeric or polymeric framework. For example, an amino acid lipid canbe attached to polyethylene glycol, polypropylene glycol, anoilgonucleotide network or lattice, a poly(amino acid), a carbohydrate,a dextran, a hydrogel, or a starch.

Amino acid lipids can be prepared as constructs attached to apharmaceutical drug compound or composition, or a biologically activeagent. For example, an amino acid lipid can be conjugated to a nucleicacid drug such as a regulatory or interfering RNA.

Examples of amino acid lipids include the following structures:

where R is any amino acid side chain.

The compounds and compositions of this disclosure may incorporatesolubilizing or functionalizing groups or structures including polymericstructures. See, e.g., R. L. Dunn and R. M. Ottenbrite, Polymeric Drugsand Drug Delivery Systems, ACS Symp. Ser. 469 (1991). Amino acid lipidscan be derivatized to enhance solubility such as, for example, to attacha diol, to prepare a quaternary ammonium or charged group, to attachhydroxyl or amine groups such as alcohols, polyols, or polyethers, or toattach a polyethyleneimine, a polyethyleneglycol or apolypropyleneglycol. The molecular mass of an attached polymericcomponent such as a polyethyleneglycol can be any value, for example,200, 300, 400, 500, 600, 750, 1000, 1250, 1500, 2000, 3000, 4000, 5000,7500, 10,000, 15,000, 20,000, 25,000, or 30,000 Da, or greater. Forexample, a polyethyleneglycol chain can be attached through an aminogroup or other reactive group of an amino acid side chain.

In general, as used herein, general chemical terms refer to all groupsof a specified type, including groups having any number and type ofatoms, unless otherwise specified. For example “alkenyl” refers broadlyto alkyls having 2 to 22 carbon atoms, as defined below, while(C18:1)alkenyl refers to alkenyls having 18 carbon atoms and one doublebond.

The term “alkyl” as used herein refers to a saturated, branched orunbranched, substituted or unsubstituted aliphatic group containing from1-22 carbon atoms. This definition applies to the alkyl portion of othergroups such as, for example, alkoxy, alkanoyl, aralkyl, and other groupsdefined below. The term “cycloalkyl” as used herein refers to asaturated, substituted or unsubstituted cyclic alkyl ring containingfrom 3 to 12 carbon atoms. As used herein, the term “C(1-5)alkyl,” forexample, includes C(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, andC(5)alkyl. Likewise, the term “C(3-22)alkyl,” for example, includesC(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, C(5)alkyl, C(6)alkyl,C(7)alkyl, C(8)alkyl, C(9)alkyl, C(10)alkyl, C(11)alkyl, C(12)alkyl,C(13)alkyl, C(14)alkyl, C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl,C(19)alkyl, C(20)alkyl, C(21)alkyl, and C(22)alkyl.

The term “alkenyl” as used herein refers to an unsaturated, branched orunbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to22 carbon atoms and at least one carbon-carbon double bond. The term“alkynyl” as used herein refers to an unsaturated, branched orunbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to22 carbon atoms and at least one carbon-carbon triple bond.

The term “alkoxy” as used herein refers to an alkyl, cycloalkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom. The term“alkanoyl” as used herein refers to —C(═O)-alkyl, which mayalternatively be referred to as “acyl.” The term “alkanoyloxy” as usedherein refers to —O—C(═O)-alkyl groups. The term “alkylamino” as usedherein refers to the group —NRR′, where R and R′ are each eitherhydrogen or alkyl, and at least one of R and R′ is alkyl. Alkylaminoincludes groups such as piperidino wherein R and R′ form a ring. Theterm “alkylaminoalkyl” refers to -alkyl-NRR′.

The term “aryl” as used herein refers to any stable monocyclic,bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in eachring, wherein at least one ring is aromatic. Some examples of an arylinclude phenyl, naphthyl, tetrahydro-naphthyl, indanyl, and biphenyl.Where an aryl substituent is bicyclic and one ring is non-aromatic, itis understood that attachment is to the aromatic ring. An aryl may besubstituted or unsubstituted.

The term “heteroaryl” as used herein refers to any stable monocyclic,bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in eachring, wherein at least one ring is aromatic and contains from 1 to 4heteroatoms selected from oxygen, nitrogen and sulfur. Some examples ofa heteroaryl include acridinyl, quinoxalinyl, pyrazolyl, indolyl,benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl,quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl,pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinolinyl. A heteroarylincludes the N-oxide derivative of a nitrogen-containing heteroaryl.

The term “heterocycle” or “heterocyclyl” as used herein refers to anaromatic or nonaromatic ring system of from five to twenty-two atoms,wherein from 1 to 4 of the ring atoms are heteroatoms selected fromoxygen, nitrogen, and sulfur. Thus, a heterocycle may be a heteroaryl ora dihydro or tetrathydro version thereof.

The term “aroyl” as used herein refers to an aryl radical derived froman aromatic carboxylic acid, such as a substituted benzoic acid. Theterm “aralkyl” as used herein refers to an aryl group bonded to an alkylgroup, for example, a benzyl group.

The term “carboxyl” as used herein represents a group of the formula—C(═O)OH or —C(═O)O⁻. The terms “carbonyl” and “acyl” as used hereinrefer to a group in which an oxygen atom is double-bonded to a carbonatom >C═O. The term “hydroxyl” as used herein refers to —OH or —O⁻. Theterm “nitrile” or “cyano” as used herein refers to —CN. The term“halogen” or “halo” refers to fluoro (—F), chloro (—Cl), bromo (—Br),and iodo (—I).

The term “substituted” as used herein refers to an atom having one ormore substitutions or substituents which can be the same or differentand may include a hydrogen substituent. Thus, the terms alkyl,cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino,alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, and aralkyl asused herein refer to groups which include substituted variations.Substituted variations include linear, branched, and cyclic variations,and groups having a substituent or substituents replacing one or morehydrogens attached to any carbon atom of the group. Substituents thatmay be attached to a carbon atom of the group include alkyl, cycloalkyl,alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino,alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl, acyl,hydroxyl, cyano, halo, haloalkyl, amino, aminoacyl, alkylaminoacyl,acyloxy, aryloxy, aryloxyalkyl, mercapto, nitro, carbamyl, carbamoyl,and heterocycle. For example, the term ethyl includes without limitation—CH₂CH₃, —CHFCH₃, —CF₂CH₃, —CHFCH₂F, —CHFCHF₂, —CHFCF₃, —CF₂CH₂F,—CF₂CHF₂, —CF₂CF₃, and other variations as described above. In general,substituents may be further substituted with any atom or group of atoms.

Amino acid lipids of this invention or variants thereof can besynthesized by methods known in the art.

Methods to prepare various organic groups and protective groups areknown in the art and their use and modification is generally within theability of one of skill in the art. See, e.g., Stanley R. Sandler andWolf Karo, Organic Functional Group Preparations (1989); Greg T.Hermanson, Bioconjugate Techniques (1996); Leroy G. Wade, Compendium OfOrganic Synthetic Methods (1980); examples of protective groups arefound in T. W. Greene and P. G. M. Wuts, Protective Groups In OrganicSynthesis (3rd ed. 1991).

A pharmaceutically acceptable salt of a peptide or protein compositionof this invention which is sufficiently basic may be an acid-additionsalt with, for example, an inorganic or organic acid such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, chlorosulfonic,trifluoroacetic, citric, maleic, acetic, propionic, oxalic, malic,maleic, malonic, fumaric, or tartaric acids, and alkane- orarenesulfonic acids such as methanesulfonic, ethanesulfonic,benzenesulfonic, chlorobenzenesulfonic, toluenesulfonic,naphthalenesulfonic, naphthalenedisulfonic, and camphorsulfonic acids.

A pharmaceutically acceptable salt of a peptide or protein compositionof this invention which is sufficiently acidic may be an alkali metalsalt, for example, a sodium or potassium salt, or an alkaline earthmetal salt, for example, a calcium or magnesium salt, or a zinc ormanganese salt, or an ammonium salt or a salt with an organic base whichprovides a physiologically-acceptable cation, for example, a salt withmethylamine, dimethylamine, trimethylamine, triethylamine, ethanolamine,diethanolamine, triethanolamine, ethylenediamine, tromethamine,N-methylglucamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine,and including salts of amino acids such as arginate, and salts oforganic acids such as glucuronic or galactunoric acids. See, forexample, Berge et al., J. Pharm. Sci. 66:1-19, 1977.

A salt or pharmaceutically-acceptable salt of a composition of thisdisclosure which contains an interfering-RNA agent and a lipid, peptide,or protein, among other components, may contain a salt complex of theinterfering-RNA agent and the lipid, peptide, or protein. A salt complexof the interfering-RNA agent and the lipid, peptide, or protein may beformed from a pharmaceutically-acceptable salt of an interfering-RNAagent, or from a pharmaceutically-acceptable salt of the lipid, peptide,or protein.

Some compounds of this disclosure may contain both basic and acidicfunctionalities that may allow the compounds to be made into either abase or acid addition salt.

Some compounds, peptides and/or protein compositions of this inventionmay have one or more chiral centers and/or geometric isomeric centers(E- and Z-isomers), and it is to be understood that the inventionencompasses all such optical isomers, diastereoisomers, geometricisomers, and mixtures thereof.

This invention encompasses any and all tautomeric, solvated orunsolvated, hydrated or unhydrated forms, as well as any atom isotopeforms of the compounds, peptides and/or protein compositions disclosedherein.

Additional Delivery Lipids

In some aspects of this invention, amino acid lipids and additionalnon-amino acid lipids may be employed for delivery and administration ofregulatory RNA components, RNA antagonists, interfering RNA, or nucleicacids. More particularly, a composition of this invention may includeone or more amino acid lipids along with non-amino acid cationic lipidsand non-amino acid non-cationic lipids.

Non-amino acid cationic lipids may be monocationic or polycationic. Somenon-amino acid cationic lipids include neutral lipids and lipids havingapproximately zero net charge at a particular pH, for example, azwitterionic lipid. Non-amino acid non-cationic lipids also includeanionic lipids.

In some embodiments, a composition is a mixture or complex of an RNAcomponent with an amino acid lipid and a non-amino acid cationic lipid.In some embodiments, a composition may be a mixture or complex of one ormore regulatory or interfering RNA agents with one or more amino acidlipids and one or more non-amino acid cationic lipids.

The compounds and compositions of this disclosure can be admixed with,or attached to various targeting ligands or agents to deliver an activeagent to a cell, tissue, organ or region of an organism. Examples oftargeting agents include antibodies, ligands for receptors, peptides,proteins, lectins, (poly)saccharides, galactose, mannose, cyclodextrins,nucleic acids, DNA, RNA, aptamers, and polyamino acids.

Examples of non-amino acid cationic lipids includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane (DOTAP),1,2-bis(dimyrstoyloxy)-3-3-(trimethylammonia)propane (DMTAP);1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE);dimethyldioctadecylammonium bromide (DDAB);3-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol);3β-[N′,N′-diguanidinoethyl-aminoethane)carbamoyl cholesterol (BGTC);2-(2-(3-(bis(3-aminopropyl)amino)propylamino)acetamido)-N,N-ditetradecylacetamide(RPR209120); pharmaceutically acceptable salts thereof, and mixturesthereof.

Examples of non-amino acid cationic lipids include1,2-dialkenoyl-sn-glycero-3-ethylphosphocholines (EPCs), such as1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine,1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, pharmaceuticallyacceptable salts thereof, and mixtures thereof.

Examples of non-amino acid cationic lipids include1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).

Examples of non-amino acid polycationic lipids includetetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleylspermine (TMTOS), tetramethlytetralauryl spermine (TMTLS),tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl spermine(TMDOS), pharmaceutically acceptable salts thereof, and mixturesthereof.

Examples of non-amino acid polycationic lipids include2,5-bis(3-aminopropylamino)-N-(2-(dioctadecylamino)-2-oxoethyl)pentanamide (DOGS);2,5-bis(3-aminopropylamino)-N-(2-(di(Z)-octadeca-9-dienylamino)-2-oxoethyl)pentanamide (DOGS-9-en);2,5-bis(3-aminopropylamino)-N-(2-(di(9Z,12Z)-octadeca-9,12-dienylamino)-2-oxoethyl)pentanamide (DLinGS);3-beta-(N⁴—(N¹,N⁸-dicarbobenzoxyspermidine)carbamoyl)cholesterol(GL-67);(9Z,9′Z)-2-(2,5-bis(3-aminopropylamino)pentanamido)propane-1,3-diyl-dioctadec-9-enoate(DOSPER);2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA); pharmaceutically acceptable salts thereof,and mixtures thereof.

Examples of non-amino acid cationic lipids include DS404-28 BGTC (CAS182056-06-0), DOSPER (CAS 178532-92-8), GL-67 (179075-30-0), RPR209120(CAS 433292-13-8), DOGS (12050-77-7), DOGS (9-en, C18:1), DLinGS(C18:2), and DOTMA (104162-48-3).

Examples of non-amino acid cationic lipids are described in U.S. Pat.Nos. 4,897,355; 5,279,833; 6,733,777; 6,376,248; 5,736,392; 5,334,761;5,459,127; 2005/0064595; 5,208,036; 5,264,618; 5,279,833; 5,283,185;5,753,613; and 5,785,992.

In some embodiments, the composition is a mixture or complex of an RNAcomponent with an amino acid lipid and a non-amino acid non-cationiclipid. In some embodiments, the composition is a mixture or complex ofone or more RNA components with one or more amino acid lipids and one ormore non-amino acid non-cationic lipids.

Non-amino acid non-cationic lipids include neutral, zwitterionic, andanionic lipids. Thus, a non-cationic zwitterionic lipid may contain acationic head group.

Examples of non-amino acid non-cationic lipids include1,2-Dilauroyl-sn-glycerol (DLG); 1,2-Dimyristoyl-sn-glycerol (DMG);1,2-Dipalmitoyl-sn-glycerol (DPG); 1,2-Distearoyl-sn-glycerol (DSG);1,2-Dilauroyl-sn-glycero-3-phosphatidic acid (sodium salt; DLPA);1,2-Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt; DMPA);1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid (sodium salt; DPPA);1,2-Distearoyl-sn-glycero-3-phosphatidic acid (sodium salt; DSPA);1,2-Diarachidoyl-sn-glycero-3-phosphocholine (DAPC);1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC);1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC);1,2-Dipalmitoyl-sn-glycero-0-ethyl-3-phosphocholine (chloride ortriflate; DPePC); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC);1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE);1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);1,2-Dilauroyl-sn-glycero-3-phosphoglycerol (sodium salt; DLPG);1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt; DMPG);1,2-Dimyristoyl-sn-glycero-3-phospho-sn-1-glycerol (ammonium salt;DMP-sn-1-G); 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (sodium salt;DPPG); 1,2-Distearoyl-sn-glycero-3-phosphoglycero (sodium salt; DSPG);1,2-Distearoyl-sn-glycero-3-phospho-sn-1-glycerol (sodium salt;DSP-sn-1-G); 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt;DPPS); 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinoPC);1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG);1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG);1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (ammonium salt; POPG);1-Palmitoyl-2-4o-sn-glycero-3-phosphocholine (P-lyso-PC);1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC); and mixturesthereof.

Examples of non-amino acid non-cationic lipids include polymericcompounds and polymer-lipid conjugates or polymeric lipids, such aspegylated lipids having PEG regions of 300, 500, 1000, 1500, 2000, 3500,or 5000 molecular weight, including polyethyleneglycols,N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(sodium salt; DMPE-MPEG-2000);N-(Carbonyl-methoxypolyethyleneglycol-5000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(sodium salt; DMPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt;DPPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt;DPPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol750)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;DSPE-MPEG-750); N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;DSPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceuticallyacceptable salts thereof, and mixtures thereof.

Examples of non-amino acid non-cationic lipids include polymeric lipidssuch as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG, anddiacylglycerol-PEG-2000 or -5000.

Examples of non-amino acid non-cationic lipids include polymeric lipidssuch as multi-branched pegylated compounds, for example DSPE-PTE020 andDSPE-AM0530K.

Examples of non-amino acid non-cationic lipids include polymeric lipidssuch as DSPE-PG8G polyglycerine lipids.

Examples of non-amino acid non-cationic lipids includedioleoylphosphatidylethanolamine (DOPE),diphytanoylphosphatidylethanolamine (DPhPE),1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC).

Examples of non-amino acid non-cationic lipids include cholesterols,sterols, and steroids such as gonanes, estranes, androstanes, pregnanes,cholanes, cholestanes, ergostanes, campestanes, poriferastanes,stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterolor zoosterol derivatives of any of the foregoing, and their biologicalintermediates and precursors, which may include, for example,cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol,zymostenol, desmosterol, 7-dehydrocholesterol, and mixtures andderivatives thereof.

Examples of non-amino acid non-cationic lipids include pegylatedcholesterols, and cholestane 3-oxo(C1-22acyl) derivatives such ascholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate,cholesteryl hexanoate, cholesteryl caprylate, cholesteryl n-decanoate,cholesteryl dodecanoate, cholesteryl myristate, cholesteryl palmitate,cholesteryl behenate, cholesteryl stearate, cholesteryl nervonate,cholesteryl pelargonate, cholesteryl n-valerate, cholesteryl oleate,cholesteryl elaidate, cholesteryl erucate, cholesteryl heptanoate,cholesteryl linolelaidate, cholesteryl linoleate, and mixtures andderivatives thereof.

Examples of non-amino acid non-cationic lipids include compounds derivedfrom plant sterols including phytosterols, beta-sitosterol, campesterol,ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol,and mixtures and derivatives thereof.

Examples of non-amino acid non-cationic lipids include bile acids,cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid,deoxycholic acid, lithocholic acid, methyl-lithocholic acid, andmixtures and derivatives thereof.

Examples of non-amino acid non-cationic lipids include compounds derivedfrom steroids including glucocorticoids, cortisol, hydrocortisone,corticosterone, Δ⁵-pregnenolone, progesterone, deoxycorticosterone,17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol,dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione,aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol,tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone,9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone,9α-fluorocortisol, and mixtures and derivatives thereof.

Examples of non-amino acid non-cationic lipids include compounds derivedfrom steroids including adrogens, testosterone, dihydrotestosterone,androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol,and mixtures and derivatives thereof.

Examples of non-amino acid non-cationic lipids include compounds derivedfrom steroids including estrogens, estriols, estrones, estradiols, andmixtures and derivatives thereof.

Examples of non-amino acid non-cationic lipids include compounds derivedfrom lumisterol and vitamin D compounds.

Examples of non-amino acid non-cationic lipids include lipids havingtails ranging from C10:0 to C22:6, for example, DDPE (C10:0) (CAS253685-27-7), DLPE (C12:0) (CAS 59752-57-7), DSPE (C18:0) (CAS1069-79-0), DOPE (C18:1) (CAS 4004-05-1), DLinPE (C18:2) (CAS20707-71-5), DLenPE (C18:3) (CAS 34813-40-6), DARAPE (C20:4) (CAS5634-86-6), DDHAPE (C22:6) (CAS 123284-81-1), DPhPE (16:0[(CH₃)₄]) (CAS201036-16-0).

Examples of non-amino acid anionic lipids include phosphatidylserine,phosphatidic acid, phosphatidylcholine, platelet-activation factor(PAF), phosphatidylethanolamine, phosphatidyl-DL-glycerol,phosphatidylinositol, phosphatidylinositol (pi(4)p, pi(4,5)p2),cardiolipin (sodium salt), lysophosphatides, hydrogenated phospholipids,sphingoplipids, gangliosides, phytosphingosine, sphinganines,pharmaceutically acceptable salts thereof, and mixtures thereof.

Uses for Regulatory RNA and RNA Interference

In some aspects, this disclosure relates generally to the fields ofregulatory RNA and RNA interference, antisense therapeutics, anddelivery of RNA therapeutics. More particularly, this invention relatesto compositions and formulations for ribonucleic acids, and their usesfor medicaments and for delivery as therapeutics. This invention relatesgenerally to methods of using ribonucleic acids in RNA interference forgene-specific inhibition of gene expression in cells, or in mammals toalter a disease state or a phenotype.

RNA interference refers to methods of sequence-specificpost-transcriptional gene silencing which is mediated by adouble-stranded RNA (dsRNA) called a short interfering RNA (siRNA). SeeFire, et al., Nature 391:806, 1998, and Hamilton, et al., Science286:950-951, 1999. RNAi is shared by diverse flora and phyla and isbelieved to be an evolutionarily-conserved cellular defense mechanismagainst the expression of foreign genes. See Fire, et al., Trends Genet.15:358, 1999.

RNAi is therefore a ubiquitous, endogenous mechanism that uses smallnoncoding RNAs to silence gene expression. See Dykxhoorn, D. M. and J.Lieberman, Annu. Rev. Biomed. Eng. 8:377-402, 2006. RNAi can regulateimportant genes involved in cell death, differentiation, anddevelopment. RNAi may also protect the genome from invading geneticelements, encoded by transposons and viruses. When a siRNA is introducedinto a cell, it binds to the endogenous RNAi machinery to disrupt theexpression of mRNA containing complementary sequences with highspecificity. Any disease-causing gene and any cell type or tissue canpotentially be targeted. This technique has been rapidly utilized forgene-function analysis and drug-target discovery and validation.Harnessing RNAi also holds great promise for therapy, althoughintroducing siRNAs into cells in vivo remains an important obstacle.

The mechanism of RNAi, although not yet fully characterized, is throughcleavage of a target mRNA. The RNAi response involves an endonucleasecomplex known as the RNA-induced silencing complex (RISC), whichmediates cleavage of a single-stranded RNA complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir, et al., Genes Dev. 15:188, 2001).

One way to carry out RNAi is to introduce or express a siRNA in cells.Another way is to make use of an endogenous ribonuclease III enzymecalled dicer. One activity of dicer is to process a long dsRNA intosiRNAs. See Hamilton, et al., Science 286:950-951, 1999; Berstein, etal., Nature 409:363, 2001. A siRNA derived from dicer is typically about21-23 nucleotides in overall length with about 19 base pairs duplexed.See Hamilton, et al., supra; Elbashir, et al., Genes Dev. 15:188, 2001.In essence, a long dsRNA can be introduced in a cell as a precursor of asiRNA.

This invention provides a range of compositions, formulations andmethods which include a regulatory RNA, an interfering nucleic acid or aprecursor thereof in combination with various components includinglipids, amino acid lipids, and natural or synthetic polymers.

The term “dsRNA” as used herein refers to any nucleic acid moleculecomprising at least one ribonucleotide molecule and capable ofinhibiting or down regulating gene expression, for example, by promotingRNA interference (“RNAi”) or gene silencing in a sequence-specificmanner. The dsRNAs of this disclosure may be suitable substrates forDicer or for association with RISC to mediate gene silencing by RNAi.One or both strands of the dsRNA can further comprise a terminalphosphate group, such as a 5′-phosphate or 5′, 3′-diphosphate. As usedherein, dsRNA molecules, in addition to at least one ribonucleotide, canfurther include substitutions, chemically-modified nucleotides, andnon-nucleotides. In certain embodiments, dsRNA molecules compriseribonucleotides up to about 100% of the nucleotide positions.

Examples of dsRNA molecules can be found in, for example, U.S. patentapplication Ser. No. 11/681,725, U.S. Pat. Nos. 7,022,828 and 7,034,009,and PCT International Application Publication No. WO/2003/070897.

Examples of modified nucleosides are found in U.S. Pat. Nos. 6,403,566,6,509,320, 6,479,463, 6,191,266, 6,083,482, 5,712,378, and 5,681,940. Amodified nucleoside may have the following structure:

wherein, X is O or CH₂, Y is O, and Z is CH₂; R₁ is selected from thegroup of adenine, cytosine, guanine, hypoxanthine, uracil, thymine, anda heterocycle wherein the heterocycle is selected from the group of asubstituted 1,3-diazine, an unsubstituted 1,3-diazine, and anunsubstituted 7H imidazo[4,5]1,3 diazine; and R₂, R₃ are independentlyselected from the group of H, OH, DMTO, TBDMSO, BnO, THPO, AcO, BzO,OP(NiPr₂)O(CH₂)₂CN, OPO₃ H, diphosphate, and triphosphate, wherein R₂and R₃ together may be PhCHO₂, TIPDSO₂ or DTBSO₂. As used herein, theabbreviation “Ac” refers to acetyl; the abbreviation “Bn” refers tobenzyl; the abbreviation “Bz” refers to benzoyl; the abbreviation “DMT”refers to dimethoxytrityl; the abbreviation “THP” refers totetrahydropyranyl; the abbreviation “TBDMS” refers tot-butyldimethylsilyl; the abbreviation “TIPDS” refers totetraisopropyldisilyl; and the abbreviation “DTBS” refers todi(t-butyl)silyl.

In addition, as used herein, the terms “dsRNA,” “RNAi-inducing agent,”and “RNAi-agent” are meant to be synonymous with other terms used todescribe nucleic acid molecules that are capable of mediating sequencespecific RNAi including meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA),gapped dsRNA (gdsRNA), short interfering nucleic acid (siRNA), siRNA,microRNA (miRNA), single strand RNA, short hairpin RNA (shRNA), shortinterfering oligonucleotide, short interfering substitutedoligonucleotide, short interfering modified oligonucleotide,chemically-modified dsRNA, and post-transcriptional gene silencing RNA(ptgsRNA), as well as precursors of any of the above.

The term “large double-stranded (ds) RNA” refers to any double-strandedRNA longer than about 40 base pairs (bp) to about 100 bp or more,particularly up to about 300 bp to about 500 bp. The sequence of a largedsRNA may represent a segment of an mRNA or an entire mRNA. Adouble-stranded structure may be formed by self-complementary nucleicacid molecule or by annealing of two or more distinct complementarynucleic acid molecule strands.

In some aspects, a dsRNA comprises two separate oligonucleotides,comprising a first strand (antisense) and a second strand (sense),wherein the antisense and sense strands are self-complementary (i.e.,each strand comprises a nucleotide sequence that is complementary to anucleotide sequence in the other strand and the two separate strandsform a duplex or double-stranded structure, for example, wherein thedouble-stranded region is about 15 to about 24 base pairs or about 26 toabout 40 base pairs); the antisense strand comprises a nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof (e.g., a human mRNA); and thesense strand comprises a nucleotide sequence corresponding (i.e.,homologous) to the target nucleic acid sequence or a portion thereof(e.g., a sense strand of about 15 to about 25 nucleotides or about 26 toabout 40 nucleotides corresponds to the target nucleic acid or a portionthereof).

In some embodiments, the dsRNA may be assembled from a singleoligonucleotide in which the self-complementary sense and antisensestrands of the dsRNA are linked by together by a nucleic acidbased-linker or a non-nucleic acid-based linker. In some embodiments,the first (antisense) and second (sense) strands of the dsRNA moleculeare covalently linked by a nucleotide or non-nucleotide linker asdescribed herein and known in the art. In some embodiments, a firstdsRNA molecule is covalently linked to at least one second dsRNAmolecule by a nucleotide or non-nucleotide linker known in the art,wherein the first dsRNA molecule can be linked to a plurality of otherdsRNA molecules that can be the same or different, or any combinationthereof. In some embodiments, the linked dsRNA may include a thirdstrand that forms a meroduplex with the linked dsRNA.

In some respects, dsRNA molecules described herein form a meroduplex RNA(mdRNA) having three or more strands, for example, an ‘A’ (first orantisense) strand, ‘S1’ (second) strand, and ‘S2’ (third) strand inwhich the ‘S1‘ and’S2’ strands are complementary to and form base pairs(bp) with non-overlapping regions of the ‘A’ strand (e.g., an mdRNA canhave the form of A: S1S2). The S1, S2, or more strands togetheressentially comprise a sense strand to the ‘A’ strand. Thedouble-stranded region formed by the annealing of the ‘S1’ and ‘A’strands is distinct from and non-overlapping with the double-strandedregion formed by the annealing of the ‘S2’ and ‘A’ strands. An mdRNAmolecule is a “gapped” molecule, meaning a “gap” ranging from 0nucleotides up to about 10 nucleotides. In some embodiments, the A:S1duplex is separated from the A:S2 duplex by a gap resulting from atleast one unpaired nucleotide (up to about 10 unpaired nucleotides) inthe ‘A’ strand that is positioned between the A:S1 duplex and the A:S2duplex and that is distinct from any one or more unpaired nucleotide atthe 3′-end of one or more of the ‘A’, ‘S1’, or ‘S2’ strands. In someembodiments, the A: S1 duplex is separated from the A:B2 duplex by a gapof zero nucleotides (i.e., a nick in which only a phosphodiester bondbetween two nucleotides is broken or missing in the polynucleotidemolecule) between the A:S1 duplex and the A:S2 duplex—which can also bereferred to as nicked dsRNA (ndsRNA). For example, A: S 1 S2 may becomprised of a dsRNA having at least two double-stranded regions thatcombined total about 14 base pairs to about 40 base pairs and thedouble-stranded regions are separated by a gap of about 0 to about 10nucleotides, optionally having blunt ends, or A: S 1S2 may comprise adsRNA having at least two double-stranded regions separated by a gap ofup to 10 nucleotides wherein at least one of the double-stranded regionscomprises between about 5 base pairs and 13 base pairs.

As described herein, a dsRNA molecule which contains three or morestrands may be referred to as a “meroduplex” RNA (mdRNA). Examples ofmdRNA molecules can be found in U.S. Provisional Patent Application Nos.60/934,930 and 60/973,398.

A dsRNA or large dsRNA may include a substitution or modification inwhich the substitution or modification may be in a phosphate backbonebond, a sugar, a base, or a nucleoside. Such nucleoside substitutionscan include natural non-standard nucleosides (e.g., 5-methyluridine or5-methylcytidine or a 2-thioribothymidine), and such backbone, sugar, ornucleoside modifications can include an alkyl or heteroatom substitutionor addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur,or other modifications known in the art.

In addition, as used herein, the term “RNAi” is meant to be equivalentto other terms used to describe sequence specific RNA interference, suchas post transcriptional gene silencing, translational inhibition, orepigenetics. For example, dsRNA molecules of this disclosure can be usedto epigenetically silence genes at the post-transcriptional level or thepre-transcriptional level or any combination thereof.

In some aspects, this invention provides compositions containing one ormore RNAi-inducing agents which are targeted to one or more genes ortarget transcripts, along with one or more delivery components. Examplesof delivery components include lipids, peptides, polymers, polymericlipids, and conjugates thereof.

The compositions and formulations of this disclosure may be used fordelivery of RNAi-inducing entities such as dsRNA, siRNA, mdRNA, miRNA,shRNA, or RNAi-inducing vectors to cells in intact mammalian subjectsincluding humans, and may also be used for delivery of these agents tocells in culture.

This disclosure also provides methods for the delivery of one or moreRNAi-inducing agents or entities to cells, organs and tissues within thebody of a mammal. In some respects, compositions containing anRNAi-inducing entity may be introduced by various routes to betransported within the body and taken up by cells in one or more organsor tissues, where expression of a target transcript is modulated.

In general, this disclosure encompasses RNAi-inducing agents that areuseful therapeutics to prevent and treat diseases or disorderscharacterized by various aberrant processes. For instance, viruses thatinfect mammals can replicate by taking control of cellular machinery ofthe host cell. See, e.g., Fields Virology (2001). Thus, dsRNAs areuseful to disrupt viral pathways which control virus production orreplication.

This disclosure includes methods for treating or preventing a viralinfection in a subject by use of one or more therapeutic RNAi-inducingagents having a broad spectrum of efficacy against strains of a targetvirus. An RNAi-inducing agent of this invention can be targeted to asequence of a viral gene in a known variant strain or variants of avirus, and exhibit sequence-specific gene silencing of the targetedviral gene in those variants. For example, an RNAi-inducing agent may betargeted to, and exhibit efficacy against a seasonal strain of influenzavirus, as well as variant strains of influenza.

Compositions and formulations of this disclosure may be used fordelivery of drug agents or biologically active agents to a variety ofcells in vitro. Examples of cells for which in vitro delivery isencompassed include epithelial cells such as A549, immortal cell linessuch as HeLa, hepatoma cells such as HepG2, rat gliosarcoma cells suchas 9 L/LacZ, human monocyte cells such as THP-1, Madin-Darby caninekidney cells (MDCK), various fibroblast cell lines, and primary cells inculture in the presence or absence of various sera, among others.

Compositions and formulations of this disclosure may be used fordelivery of drug agents or biologically active agents to a variety ofcells, tissues or organs in vivo. Modalities for delivering an agent invivo include topical, enteral, and parenteral routes. Examples ofmodalities for delivering an agent in vivo include inhalation ofparticles or droplets, delivery of nasal or nasal-pharngyl drops,particles, or suspensions, transdermal and transmucosal routes, as wellas injection or infusion by intramuscular, subcutaneous, intravenous,intraarterial, intracardiac, intrathecal, intraosseus, intraperitoneal,and epidural routes.

In some embodiments, an agent can be administered ex vivo by directexposure to cells, tissues or organs originating from a mammaliansubject.

A drug agent or biologically active agent to be delivered using acomposition or formulation of this disclosure may be found in any formincluding, for example, a pure form, a crystalline form, a solid form, ananoparticle, a condensed form, a complexed form, or a conjugated form.

This invention also provides methods for the delivery of one or moreRNAi-inducing entities to organs and tissues within the body of amammal. In some embodiments, compositions containing an RNAi-inducingentity, one or more amino acid lipids, and one or more additional lipidcomponents are introduced by various routes to be transported within thebody and taken up by cells in one or more organs or tissues, whereexpression of a target transcript is modulated.

This disclosure provides pharmaceutically acceptable nucleic acidcompositions with various lipids useful for therapeutic delivery ofnucleic acids and gene-silencing RNAs. In particular, this inventionprovides compositions and methods for in vitro and in vivo delivery ofdsRNAs for decreasing, downregulating, or silencing the translation of atarget nucleic acid sequence or expression of a gene. These compositionsand methods may be used for prevention and/or treatment of diseases in amammal. In exemplary methods of this invention, a ribonucleic acidmolecule such as an siRNA or shRNA is contacted with an amino acid lipidto formulate a composition which can be administered to cells orsubjects such as mammals. In some embodiments, this invention providesmethods for delivering an siRNA or shRNA intracellularly by contacting anucleic acid-containing composition with a cell.

In exemplary embodiments, this invention includes compositionscontaining a nucleic acid molecule, such as a double-stranded RNA(dsRNA), a short interfering RNA (siRNA), or a short hairpin RNA(shRNA), admixed or complexed with an amino acid lipid, and a polymericlipid to form a composition that enhances intracellular delivery of thenucleic acid molecule. In some embodiments, a delivery composition ofthis invention may contain a dsRNA and one, two, or more amino acidlipids, which may be cationic or non-cationic. In some variations, adelivery composition may contain a dsRNA, amino acid lipids, and one ormore polymeric lipids. In some embodiments, a delivery composition maycontain a dsRNA, amino acid lipids, one or more additional lipids, andone or more polymeric lipids. The compositions of this invention canform stable particles which may incorporate a dsRNA as an interferingRNA agent. Compositions and formulations of this invention may includefurther delivery-enhancing components or excipients.

In some embodiments, compositions of this invention contain stableRNA-lipid particles having diameters from about 5 nm to about 400 nm. Insome embodiments, the particles may have a uniform diameter of fromabout 10 nm to about 300 nm. In some embodiments, the particles may havea uniform diameter of from about 50 nm to about 150 nm.

Within exemplary compositions of this invention, a double-stranded RNAmay be admixed or complexed with amino acid lipids to form a compositionthat enhances intracellular delivery of the dsRNA as compared tocontacting target cells with naked dsRNA.

In some embodiments, a composition of this invention may contain one ormore amino acid lipids which are from about 0.5% to about 70% (mol %) ofthe total amount of lipid and delivery-enhancing components, includingany polymeric component, but not including the RNA component. In someembodiments, a composition of this invention may contain one or moreamino acid lipids from about 10% to about 55%. In some embodiments, acomposition of this invention may contain one or more amino acid lipidsfrom about 15% to about 35%.

In certain embodiments, a composition of this invention may contain oneor more non-amino acid non-cationic lipids, where the non-amino acidnon-cationic lipids are from about 2% to about 95% (mol %) of the totalamount of lipid and delivery-enhancing components, including anypolymeric component, but not including the RNA component. In someembodiments, a composition of this invention may contain one or morenon-cationic lipids from about 20% to about 75%, or from about 45% toabout 75%, or from about 45% to about 55%. In some embodiments, acomposition of this invention may contain one or more non-cationiclipids from about 10% to about 50%.

In some embodiments, a composition of this invention may contain one ormore polymeric lipids, where the polymeric lipids are from about 0.2% toabout 20% (mol %) of the total amount of lipid and delivery-enhancingcomponents, including any polymeric component, but not including the RNAcomponent. In some embodiments, a composition of this invention maycontain one or more polymeric lipids from about 0.5% to about 10%. Insome embodiments, a composition of this invention may contain one ormore polymeric lipids from about 1% to about 5% of the composition.

Compositions and Uses for Nucleic Acid Therapeutics

In some embodiments, this invention provides a method of treating adisease or disorder in a mammalian subject. A therapeutically effectiveamount of a composition of this invention containing an interfering RNA,an amino acid lipid, a non-amino acid non-cationic lipid, a polymericlipid, and one or more delivery-enhancing components or excipients maybe administered to a subject having a disease or disorder associatedwith expression or overexpression of a gene that can be reduced,decreased, downregulated, or silenced by the composition.

This invention encompasses methods for treating a disease of the lungsuch as respiratory distress, asthma, cystic fibrosis, pulmonaryfibrosis, chronic obstructive pulmonary disease, bronchitis, oremphysema, by administering to the subject a therapeutically effectiveamount of a composition.

This invention encompasses methods for treating rheumatoid arthritis,liver disease, encephalitis, bone fracture, heart disease, viral diseaseincluding hepatitis and influenza, or cancer.

Methods for making liposomes are given in, for example, G. Gregoriadis,Liposome Technology (CRC Press 1984), M. J. Ostro, Liposomes (MarcelDekker 1987); Subhash C. Basu and Manju Basu, Liposome Methods andProtocols (2002).

The nucleic acid component, amino acid lipids, and any additionalcomponents may be mixed together first in a suitable medium such as acell culture medium, after which one or more additional lipids orcompounds may be added to the mixture. Alternatively, the amino acidlipids can be mixed together first in a suitable medium such as a cellculture medium, after which the nucleic acid component can be added.

Within certain embodiments of the invention, a dsRNA is admixed with oneor more amino acid lipids, or a combination of one or more amino acidlipids and non-amino acid non-cationic lipids.

In some embodiments, an amino acid lipid transfection/deliveryformulation can be prepared by rehydration of a dried preparation. Thelipids may be solubilized in CHCl₃, dried under nitrogen, and rehydratedin 10 mM HEPES, 5% dextrose at pH 7.4, for example, and sonicated toform liposomes. Liposomes can be diluted in HEPES dextrose. The dsRNAmay be diluted in 10 mM HEPES, 5% dextrose, pH 7.4, at 0.0008 μmol/mL.One volume of liposome can be added to one volume dsRNA and vortexed(self assembly), providing a final dsRNA concentration of 100 nM to 6.25nM dsRNA. This mixture can be diluted one to four in the presence ofcell culture media.

The interfering RNA agent may also be complexed with, or conjugated toan amino acid lipid or polymeric lipid, and admixed with one or morenon-amino acid non-cationic lipids, or a combination of one or morenon-amino acid non-cationic and non-amino acid cationic lipids.

An interfering RNA agent and an amino acid lipid may be mixed togetherfirst, followed by the addition of one or more non-amino acidnon-cationic lipids, or a combination of non-amino acid non-cationic andnon-amino acid cationic lipids added in a suitable medium such as a cellculture medium. Alternatively, the amino acid lipids and non-amino acidlipid components may be mixed first, followed by the addition of the RNAagent in a suitable medium.

In some embodiments, this disclosure includes micellar dispersioncompositions containing a drug or active agent admixed or complexed withan amino acid lipid and a dispersant to form a composition that providesintracellular delivery of the drug or active agent.

In certain embodiments, a dispersion composition of this disclosure maycontain one or more drugs or active agents, one or more amino acidlipids, and one or more dispersants. In some variations, a deliverycomposition may contain a drug or active agent, a dispersant, an aminoacid lipid, and an optional polymeric lipid. The dispersion compositionsof this disclosure can form stable particles which may incorporate thedrug or active agent.

In some aspects, a dispersion composition of this disclosure may containstable nucleic acid dispersion particles having diameters from about 5nm to about 400 nm. In some embodiments, the particles may have auniform diameter of from about 10 nm to about 300 nm. In someembodiments, the particles may have a uniform diameter of from about 50nm to about 150 nm.

A micellar dispersion can be used to formulate and improve thebioavailability of a drug or active agent, including RNAi therapeutics.While a conventional lipid-drug complex may contain a lipid bilayer orliposomal structure which maintains a hydrophilic or aqueous core, amicellar dispersion can provide dispersion droplets or nanoparticleshaving a hydrophobic oil-like core. The dispersion nanoparticles can besuspended in a continuous aqueous phase. A dispersion structure canavoid some disadvantages inherent in using a liposomal structure fordelivery of active agents, and can provide advantages in deliverybecause of the lipophilic core.

This disclosure provides a range of micellar dispersion compositionscontaining lipids and dispersants for drugs or medicaments, and fordelivery and administration of RNA agents.

Examples of dispersants include synthetic compounds includingpolyoxyglycerides such as polyglycolated capryl glycerides, ethoxydiglycol, pegylated fatty glycerides, diethylene glycol monoethylethers, and mixtures thereof. Examples of dispersants include LABRAFIL,LABRASOL, ARLATONE, TRANSCUTOL, and mixtures thereof. Examples ofdispersants include synthetic compounds such asalkylphospho-N-methylethanolamines and alkoylsarcosines. Examples ofdispersants include FOS-MEA and CRODASINIC.

In some embodiments, a delivery composition of this disclosure maycontain a drug or active agent, one or more oils, one or more amino acidlipids, and emulsifier and stabilizer lipids. In some variations, adelivery composition may contain a drug or active agent, an oil, a lipidemulsifier, an amino acid lipid, a non-cationic lipid, and a polymericlipid.

The compositions of this disclosure can form stable particles which mayincorporate a drug or active agent. In some aspects, compositions ofthis disclosure contain stable drug or active agent emulsion particleshaving diameters from about 5 nm to about 400 nm. In some embodiments,the particles may have a uniform diameter of from about 10 nm to about300 nm. In some embodiments, the particles may have a uniform diameterof from about 50 nm to about 150 nm.

Within exemplary compositions of this disclosure, a drug or active agentmay be admixed or complexed with an oil, an emulsifier, an amino acidlipid, and a polymeric stabilizing lipid, to form a composition thatenhances intracellular delivery of the drug or active agent.

An oil-in-water emulsion can be used to formulate and improve thebioavailability of a drug or active agent, including RNAi therapeutics.

While a conventional lipid-drug complex may contain a lipid bilayer orliposomal structure which maintains a hydrophilic or aqueous core, anoil-in-water emulsion can provide emulsion droplets or nanoparticleshaving a lipid layer surrounding a hydrophobic oil core. The emulsiondroplets or nanoparticles can be suspended in a continuous aqueousphase. An emulsion structure can avoid some disadvantages inherent inusing a liposomal structure for delivery of active agents, and canprovide advantages in delivery because of the lipophilic core.

A range of novel emulsion compositions are provided in this disclosureincluding novel compositions and uses of oils, emulsifiers, and lipidcomponents with interfering-RNA agents.

Examples of oils include synthetic oils, fatty acid esters of propyleneglycols, ethers of ethylene glycols, glyceryl oils, cholesteryl oils,vegetable oils, nut oils, essential oils, mineral oil, lipid-solublecompounds such as tocopherols and Vitamin E, and mixtures thereof.Examples of oils include synthetic oils such as CAPRYOL 90 (propyleneglycol monoester), CAPRYOL PGMC (propylene glycol monoester), LABRAFACPC (propylene glycol monoester), LABRAFAC PG (propylene glycol diester),LAUROGLYCOL 90 (propylene glycol monoester), LAUROGLYCOL FCC (propyleneglycol monoester), PLUROL OLEIQUE CC 497 (propylene glycol monoester),LABRAFAC LIPOPHILE WL 1349 (triglyceride), PECEOL (glyceryl monoester),MAISINE 35-1 (glyceryl monoester), and mixtures thereof.

Compositions and Methods for RNA Therapeutics

This invention provides compositions and methods for modulating geneexpression using regulatory RNA such as by RNA interference. Acomposition of this invention can deliver a ribonucleic acid agent to acell which can produce the response of RNAi. Examples of nucleic acidagents useful for this invention include double-stranded nucleic acids,modified or degradation-resistant nucleic acids, RNA, siRNA, siRNA,shRNA, miRNA, piRNA, RNA antagonists, single-stranded nucleic acids,DNA-RNA chimeras, antisense nucleic acids, and ribozymes. As usedherein, the terms siRNA, siRNA, and shRNA include precursors of siRNA,siRNA, and shRNA, respectively. For example, the term siRNA includes anRNA or double-stranded RNA that is suitable as a substrate of dicerenzyme.

Ribonucleic acid agents useful for this invention may be targeted tovarious genes. Examples of human genes suitable as targets include TNF,FLT1, the VEGF family, the ERBB family, the PDGFR family, BCR-ABL, andthe MAPK family, among others. Examples of human genes suitable astargets and nucleic acid sequences thereto include those disclosed inPCT/US08/55333, PCT/US08/55339, PCT/US08/55340, PCT/US08/55341,PCT/US08/55350, PCT/US08/55353, PCT/US08/55356, PCT/US08/55357,PCT/US08/55360, PCT/US08/55362, PCT/US08/55365, PCT/US08/55366,PCT/US08/55369, PCT/US08/55370, PCT/US08/55371, PCT/US08/55372,PCT/US08/55373, PCT/US08/55374, PCT/US08/55375, PCT/US08/55376,PCT/US08/55377, PCT/US08/55378, PCT/US08/55380, PCT/US08/55381,PCT/US08/55382, PCT/US08/55383, PCT/US08/55385, PCT/US08/55386,PCT/US08/55505, PCT/US08/55511, PCT/US08/55515, PCT/US08/55516,PCT/US08/55519, PCT/US08/55524, PCT/US08/55526, PCT/US08/55527,PCT/US08/55532, PCT/US08/55533, PCT/US08/55542, PCT/US08/55548,PCT/US08/55550, PCT/US08/55551, PCT/US08/55554, PCT/US08/55556,PCT/US08/55560, PCT/US08/55563, PCT/US08/55597, PCT/US08/55599,PCT/US08/55601, PCT/US08/55603, PCT/US08/55604, PCT/US08/55606,PCT/US08/55608, PCT/US08/55611, PCT/US08/55612, PCT/US08/55615,PCT/US08/55618, PCT/US08/55622, PCT/US08/55625, PCT/US08/55627,PCT/US08/55631, PCT/US08/55635, PCT/US08/55644, PCT/US08/55649,PCT/US08/55651, PCT/US08/55662, PCT/US08/55672, PCT/US08/55676,PCT/US08/55678, PCT/US08/55695, PCT/US08/55697, PCT/US08/55698,PCT/US08/55701, PCT/US08/55704, PCT/US08/55708, PCT/US08/55709, andPCT/US08/55711.

An RNA of this disclosure to be delivered may have a sequence that iscomplementary to a region of a viral gene. For example, somecompositions and methods of this invention are useful to regulateexpression of the viral genome of an influenza virus. In someembodiments, this invention provides compositions and methods formodulating expression and infectious activity of an influenza by RNAinterference. Expression and/or activity of an influenza can bemodulated by delivering to a cell, for example, a short interfering RNAmolecule having a sequence that is complementary to a region of a RNApolymerase subunit of an influenza. Examples of RNAs targeted to aninfluenza virus are given in U.S. Patent Publication No. 20070213293 A1.

In some embodiments, this invention provides compositions and methodsfor inhibiting expression of a target transcript in a subject byadministering to the subject a composition containing an effectiveamount of an RNAi-inducing compound such as a short interferingoligonucleotide molecule, or a precursor thereof. RNAi uses smallinterfering RNAs (siRNAs) to target messenger RNA (mRNAs) and attenuatetranslation. A siRNA as used in this invention may be a precursor fordicer processing such as, for example, a long dsRNA processed into asiRNA. This invention provides methods of treating or preventingdiseases or conditions associated with expression of a target transcriptor activity of a peptide or protein encoded by the target transcript.

A therapeutic strategy based on RNAi can be used to treat a wide rangeof diseases by shutting down the growth or function of a virus ormicroorganism, as well as by shutting down the function of an endogenousgene product in the pathway of the disease.

In some embodiments, this invention provides novel compositions andmethods for delivery of RNAi-inducing entities such as short interferingoligonucleotide molecules, and precursors thereof. In particular, thisinvention provides compositions containing an RNAi-inducing entity whichis targeted to one or more transcripts of a cell, tissue, and/or organof a subject.

A siRNA can be two RNA strands having a region of complementarity about19 nucleotides in length. A siRNA optionally includes one or twosingle-stranded overhangs or loops.

A shRNA can be a single RNA strand having a region ofself-complementarity. The single RNA strand may form a hairpin structurewith a stem and loop and, optionally, one or more unpaired portions atthe 5′ and/or 3′ portion of the RNA.

The active therapeutic agent can be a chemically-modified RNA withimproved resistance to nuclease degradation in vivo, and/or improvedcellular uptake, which retains RNAi activity.

A siRNA agent of this invention may have a sequence that iscomplementary to a region of a target gene. A siRNA of this inventionmay have 29-50 base pairs, for example, a dsRNA having a sequence thatis complementary to a region of a target gene. Alternately, thedouble-stranded nucleic acid can be a dsDNA.

In certain embodiments, the active agent can be a short interferingnucleic acid (siRNA), short interfering RNA (siRNA), double-stranded RNA(dsRNA), micro-RNA, or short hairpin RNA (shRNA) that can modulateexpression of a gene product.

Comparable methods and compositions are provided that target expressionof one or more different genes associated with a particular diseasecondition in a subject, including any of a large number of genes whoseexpression is known to be aberrantly increased as a causal orcontributing factor associated with the selected disease condition.

The RNAi-inducing compound of this invention can be administered inconjunction with other known treatments for a disease condition.

In some embodiments, this invention features compositions containing asmall nucleic acid molecule, such as short interfering nucleic acid, ashort interfering RNA, a double-stranded RNA, a micro-RNA, or a shorthairpin RNA, admixed or complexed with, or conjugated to, adelivery-enhancing compound.

As used herein, the terms “regulatory RNA,” “short interfering nucleicacid,” “siRNA,” “short interfering RNA,” “short interferingoligonucleotide molecule,” and “chemically-modified short interferingnucleic acid molecule,” refer to any nucleic acid molecule capable ofregulating, inhibiting or down regulating gene expression or, forexample, viral replication, by mediating RNA interference (RNAi) or genesilencing in a sequence-specific manner. Regulatory RNA includessingle-stranded RNA antagonists.

In some embodiments, the siRNA is a double-stranded polynucleotidemolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in a target ribonucleic acidmolecule for down regulating expression, or a portion thereof, and thesense region comprises a nucleotide sequence corresponding to (i.e.,which is substantially identical in sequence to) the target ribonucleicacid sequence or portion thereof.

As used herein, “siRNA” means a small interfering ribonucleic acid thatis a relatively short-length double-stranded nucleic acid, or optionallya longer precursor thereof. The length of useful siRNAs within thisinvention will in some embodiments be preferred at a length ofapproximately 20 to 50 bp. However, there is no particular limitation tothe length of useful siRNAs, including siRNAs. For example, siRNAs caninitially be presented to cells in a precursor form that issubstantially different than a final or processed form of the siRNA thatwill exist and exert gene silencing activity upon delivery, or afterdelivery, to the target cell. Precursor forms of siRNAs may, forexample, include precursor sequence elements that are processed,degraded, altered, or cleaved at or after the time of delivery to yielda siRNA that is active within the cell to mediate gene silencing. Insome embodiments, useful siRNAs will have a precursor length, forexample, of approximately 100-200 base pairs, or 50-100 base pairs, orless than about 50 base pairs, which will yield an active, processedsiRNA within the target cell. In other embodiments, a useful siRNA orsiRNA precursor will be approximately 10 to 49 bp, or 15 to 35 bp, orabout 21 to 30 bp in length.

In certain embodiments of this invention, polynucleotidedelivery-enhancing polypeptides may be used to facilitate delivery ofnucleic acid molecules, including large nucleic acid precursors ofsiRNAs. For example, the methods and compositions herein may be employedfor enhancing delivery of larger nucleic acids that represent“precursors” to desired siRNAs, wherein the precursor amino acids may becleaved or otherwise processed before, during or after delivery to atarget cell to form an active siRNA for modulating gene expressionwithin the target cell.

For example, a dsRNA precursor polynucleotide may be selected as acircular, single-stranded polynucleotide, having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof, and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active dsRNA molecule capable ofinducing RNAi.

siRNA molecules of this invention, particularly non-precursor forms, canbe less than 30 base pairs, or about 17-19 bp, or 19-21 bp, or 21-23 bp.

siRNAs can mediate selective gene silencing in the mammalian system.Hairpin RNAs, with a short loop and 19 to 27 base pairs in the stem,also selectively silence expression of genes that are homologous to thesequence in the double-stranded stem. Mammalian cells can convert shorthairpin RNA into siRNA to mediate selective gene silencing.

RISC mediates cleavage of single stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place within the region complementary to theantisense strand of the siRNA duplex. siRNA duplexes of 21 nucleotidesare typically most active when containing two-nucleotide 3′-overhangs.

Replacing the 3′-overhanging segments of a 21-mer siRNA duplex having2-nucleotide 3′ overhangs with deoxyribonucleotides may not have anadverse effect on RNAi activity. Replacing up to 4 nucleotides on eachend of the siRNA with deoxyribonucleotides can be tolerated.

Alternatively, siRNAs can be delivered as single or multipletranscription products expressed by a polynucleotide vector encoding thesingle or multiple siRNAs and directing their expression within targetcells. In these embodiments the double-stranded portion of a finaltranscription product of the siRNAs to be expressed within the targetcell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bplong.

In some embodiments of this invention, the double-stranded region ofsiRNAs in which two strands are paired may contain bulge or mismatchedportions, or both. Double-stranded portions of siRNAs in which twostrands are paired are not limited to completely paired nucleotidesegments, and may contain nonpairing portions due to, for example,mismatch (the corresponding nucleotides not being complementary), bulge(lacking in the corresponding complementary nucleotide on one strand),or overhang. Nonpairing portions can be contained to the extent thatthey do not interfere with siRNA formation. In some embodiments, a“bulge” may comprise 1 to 2 nonpairing nucleotides, and thedouble-stranded region of siRNAs in which two strands pair up maycontain from about 1 to 7, or about 1 to 5 bulges. In addition,“mismatch” portions contained in the double-stranded region of siRNAsmay be present in numbers from about 1 to 7, or about 1 to 5. Most oftenin the case of mismatches, one of the nucleotides is guanine, and theother is uracil. Such mismatching may be attributable, for example, to amutation from C to T, G to A, or mixtures thereof, in a correspondingDNA coding for sense RNA, but other causes are also contemplated.

The terminal structure of siRNAs of this invention may be either bluntor cohesive (overhanging) as long as the siRNA retains its activity tosilence expression of target genes. The cohesive (overhanging) endstructure is not limited to the 3′ overhang, but includes the 5′overhanging structure as long as it retains activity for inducing genesilencing. In addition, the number of overhanging nucleotides is notlimited to 2 or 3 nucleotides, but can be any number of nucleotides aslong as it retains activity for inducing gene silencing. For example,overhangs may comprise from 1 to about 8 nucleotides, or from 2 to 4nucleotides.

The length of siRNAs having overhang end structure may be expressed interms of the paired duplex portion and any overhanging portion at eachend. For example, a 25/27-mer siRNA duplex with a 2-bp 3′ antisenseoverhang has a 25-mer sense strand and a 27-mer antisense strand, wherethe paired portion has a length of 25 bp.

Any overhang sequence may have low specificity to a target gene, and maynot be complementary (antisense) or identical (sense) to the target genesequence. As long as the siRNA retains activity for gene silencing, itmay contain in the overhang portion a low molecular weight structure,for example, a natural RNA molecule such as a tRNA, an rRNA, a viralRNA, or an artificial RNA molecule.

The terminal structure of the siRNAs may have a stem-loop structure inwhich ends of one side of the double-stranded nucleic acid are connectedby a linker nucleic acid, for example, a linker RNA. The length of thedouble-stranded region (stem portion) can be, for example, 15 to 49 bp,or 15 to 35 bp, or about 21 to 30 bp long. Alternatively, the length ofthe double-stranded region that is a final transcription product ofsiRNAs to be expressed in a target cell may be, for example,approximately 15 to 49 bp, or 15 to 35 bp, or about 21 to 30 bp long.

The siRNA can contain a single stranded polynucleotide having anucleotide sequence complementary to a nucleotide sequence in a targetnucleic acid molecule, or a portion thereof, wherein the single strandedpolynucleotide can contain a terminal phosphate group, such as a5′-phosphate (see e.g. Martinez, et al., Cell. 110:563-574, 2002, andSchwarz, et al., Molecular Cell 10:537-568, 2002, or 5′,3′-diphosphate.

As used herein, the term siRNA is not limited to molecules containingonly naturally-occurring RNA or DNA, but also encompasseschemically-modified nucleotides and non-nucleotides. In someembodiments, the short interfering nucleic acid molecules of theinvention lack 2′-hydroxy (2′-OH) containing nucleotides. In someembodiments, short interfering nucleic acids do not require the presenceof nucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of this invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′—OHgroup). siRNA molecules that do not require the presence ofribonucleotides within the siRNA molecule to support RNAi can, however,have an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′—OH groups. siRNA molecules can comprise ribonucleotides in at leastabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions.

As used herein, the term siRNA encompasses nucleic acid molecules thatare capable of mediating sequence specific RNAi such as, for example,short interfering RNA (siRNA) molecules, double-stranded RNA (dsRNA)molecules, micro-RNA molecules, short hairpin RNA (shRNA) molecules,short interfering oligonucleotide molecules, short interfering nucleicacid molecules, short interfering modified oligonucleotide molecules,chemically-modified siRNA molecules, and post-transcriptional genesilencing RNA (ptgsRNA) molecules, among others.

In some embodiments, siRNA molecules comprise separate sense andantisense sequences or regions, wherein the sense and antisense regionsare covalently linked by nucleotide or non-nucleotide linker molecules,or are non-covalently linked by ionic interactions, hydrogen bonding,van der waals interactions, hydrophobic interactions, and/or stackinginteractions.

“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene mRNA, that can induce RNAi by binding to the target genemRNA.

“Sense RNA” is an RNA strand having a sequence complementary to anantisense RNA, and anneals to its complementary antisense RNA to form asiRNA.

As used herein, the term “RNAi construct” or “RNAi precursor” refers toan RNAi-inducing compound such as small interfering RNAs (siRNAs),hairpin RNAs, and other RNA species which can be cleaved in vivo to forma siRNA. RNAi precursors herein also include expression vectors (alsoreferred to as RNAi expression vectors) capable of giving rise totranscripts which form dsRNAs or hairpin RNAs in cells, and/ortranscripts which can produce siRNAs in vivo.

A siHybrid molecule is a double-stranded nucleic acid that has a similarfunction to siRNA. Instead of a double-stranded RNA molecule, a siHybridis comprised of an RNA strand and a DNA strand. Preferably, the RNAstrand is the antisense strand which binds to a target mRNA. ThesiHybrid created by the hybridization of the DNA and RNA strands have ahybridized complementary portion and preferably at least one 3′overhanging end.

siRNAs for use within the invention can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e., each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs). The antisense strand may comprise a nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof, and the sense strand maycomprise a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siRNA can be assembledfrom a single oligonucleotide, where the self-complementary sense andantisense regions of the siRNA are linked by means of a nucleicacid-based or non-nucleic acid-based linker(s).

In some embodiments, siRNAs for intracellular delivery can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof, and the sense regioncomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof.

Examples of chemical modifications that can be made in an siRNA includephosphorothioate internucleotide linkages, 2′-deoxyribonucleotides,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation.

The antisense region of a siRNA molecule can include a phosphorothioateinternucleotide linkage at the 3′-end of said antisense region. Theantisense region can comprise about one to about five phosphorothioateinternucleotide linkages at the 5′-end of said antisense region. The3′-terminal nucleotide overhangs of a siRNA molecule can includeribonucleotides or deoxyribonucleotides that are chemically-modified ata nucleic acid sugar, base, or backbone. The 3′-terminal nucleotideoverhangs can include one or more universal base ribonucleotides. The3′-terminal nucleotide overhangs can comprise one or more acyclicnucleotides.

For example, a chemically-modified siRNA can have 1, 2, 3, 4, 5, 6, 7,8, or more phosphorothioate internucleotide linkages in one strand, orcan have 1 to 8 or more phosphorothioate internucleotide linkages ineach strand. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siRNA duplex, forexample in the sense strand, the antisense strand, or both strands.

siRNA molecules can comprise one or more phosphorothioateinternucleotide linkages at the 3′-end, the 5′-end, or both of the 3′-and 5′-ends of the sense strand, the antisense strand, or in bothstrands. For example, an exemplary siRNA molecule can include 1, 2, 3,4, 5, or more consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.

In certain embodiments, a siRNA molecule includes 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more pyrimidine phosphorothioate internucleotide linkagesin the sense strand, the antisense strand, or in both strands.

In some embodiments, a siRNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more purine phosphorothioate internucleotide linkages in thesense strand, the antisense strand, or in both strands.

A siRNA molecule can include a circular nucleic acid molecule, whereinthe siRNA is about 38 to about 70, for example, about 38, 40, 45, 50,55, 60, 65, or 70 nucleotides in length, having about 18 to about 23,for example, about 18, 19, 20, 21, 22, or 23 base pairs, wherein thecircular oligonucleotide forms a dumbbell-shaped structure having about19 base pairs and 2 loops.

A circular siRNA molecule can contain two loop motifs, wherein one orboth loop portions of the siRNA molecule is biodegradable. For example,the loop portions of a circular siRNA molecule may be transformed invivo to generate a double-stranded siRNA molecule with 3′-terminaloverhangs, such as 3′-terminal nucleotide overhangs comprising about 2nucleotides.

Modified nucleotides in a siRNA molecule can be in the antisense strand,the sense strand, or both. For example, modified nucleotides can have aNorthern conformation (e.g., Northern pseudorotation cycle; see e.g.,Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed.,1984). Examples of nucleotides having a Northern configuration includelocked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethoxy (MOE)nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides.

Chemically modified nucleotides can be resistant to nuclease degradationwhile at the same time maintaining the capacity to mediate RNAi.

The sense strand of a double stranded siRNA molecule may have a terminalcap moiety such as an inverted deoxyabasic moiety, at the 3′-end,5′-end, or both 3′ and 5′-ends of the sense strand.

Examples of conjugates include conjugates and ligands described inVargeese, et al., U.S. application Ser. No. 10/427,160, filed Apr. 30,2003, incorporated by reference herein in its entirety, including thedrawings.

In some embodiments of this invention, the conjugate may be covalentlyattached to the chemically-modified siRNA molecule via a biodegradablelinker. For example, the conjugate molecule may be attached at the3′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siRNA molecule.

In certain embodiments, the conjugate molecule is attached at the 5′-endof either the sense strand, the antisense strand, or both strands of thechemically-modified siRNA molecule. In some embodiments, the conjugatemolecule is attached both the 3′-end and 5′-end of either the sensestrand, the antisense strand, or both strands of the chemically-modifiedsiRNA molecule, or any combination thereof.

In some embodiments, a conjugate molecule comprises a molecule thatfacilitates delivery of a chemically-modified siRNA molecule into abiological system, such as a cell.

In some embodiments, a conjugate molecule attached to thechemically-modified siRNA molecule is a polyethylene glycol, human serumalbumin, or a ligand for a cellular receptor that can mediate cellularuptake. Examples of specific conjugate molecules contemplated by theinstant invention that can be attached to chemically-modified siRNAmolecules are described in Vargeese, et al., U.S. Patent PublicationNos. 20030130186 and 20040110296.

A siRNA may be contain a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of thesiRNA to the antisense region of the siRNA. In some embodiments, anucleotide linker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength. In some embodiments, the nucleotide linker can be a nucleic acidaptamer. As used herein, the terms “aptamer” or “nucleic acid aptamer”encompass a nucleic acid molecule that binds specifically to a targetmolecule, wherein the nucleic acid molecule contains a sequence that isrecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid.

For example, the aptamer can be used to bind to a ligand-binding domainof a protein, thereby preventing interaction of the naturally occurringligand with the protein. See, for example, Gold, et al., Annu. Rev.Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun,Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000;Hermann and Patel, Science 287:820, 2000; and Jayasena, ClinicalChemistry 45:1628, 1999.

A non-nucleotide linker can be an abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, orother polymeric compounds (e.g., polyethylene glycols such as thosehaving between 2 and 100 ethylene glycol units). Specific examplesinclude those described by Seela and Kaiser, Nucleic Acids Res. 18:6353,1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am.Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J Am. Chem. Soc.113:5109, 1991; Ma, et al., Nucleic Acids Res. 21:2585, 1993, andBiochemistry 32:1751, 1993; Durand, et al., Nucleic Acids Res. 18:6353,1990; McCurdy, et al., Nucleosides & Nucleotides 10:287, 1991; Jaschke,et al., Tetrahedron Lett. 34:301-304, 1993; Ono, et al., Biochemistry30:9914, 1991; Arnold, et al., International Publication No. WO89/02439; Usman, et al., International Publication No. WO 95/06731;Dudycz, et al., International Publication No. WO 95/11910, and Ferentzand Verdine, J. Am. Chem. Soc. 113:4000, 1991.

A “non-nucleotide linker” refers to a group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine, for example at the C1 position of the sugar.

In some embodiments, modified siRNA molecule can have phosphate backbonemodifications including one or more phosphorothioate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions. Examples of oligonucleotide backbone modifications aregiven in Hunziker and Leumann, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, pp. 331-417, 1995, andMesmaeker, et al., Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, pp. 24-39, 1994.

siRNA molecules, which can be chemically-modified, can be synthesizedby: (a) synthesis of two complementary strands of the siRNA molecule;and (b) annealing the two complementary strands together underconditions suitable to obtain a double-stranded siRNA molecule. In someembodiments, synthesis of the complementary portions of the siRNAmolecule is by solid phase oligonucleotide synthesis, or by solid phasetandem oligonucleotide synthesis.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example, as described in Caruthers, etal., Methods in Enzymology 211:3-19, 1992; Thompson, et al.,International PCT Publication No. WO 99/54459; Wincott, et al., NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59,1997; Brennan, et al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan,U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain siRNAmolecules of the invention, follows general procedures as described, forexample, in Usman, et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe,et al., Nucleic Acids Res. 18:5433, 1990; and Wincott, et al., NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59,1997.

An “asymmetric hairpin” as used herein is a linear siRNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop.

An “asymmetric duplex” as used herein is a siRNA molecule having twoseparate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.

To “modulate gene expression” as used herein is to upregulate ordownregulate expression of a target gene, which can include upregulationor downregulation of mRNA levels present in a cell, or of mRNAtranslation, or of synthesis of protein or protein subunits, encoded bythe target gene.

The terms “inhibit,” “down-regulate,” or “reduce expression,” as usedherein mean that the expression of the gene, or level of RNA moleculesor equivalent RNA molecules encoding one or more proteins or proteinsubunits, or level or activity of one or more proteins or proteinsubunits encoded by a target gene, is reduced below that observed in theabsence of the nucleic acid molecules (e.g., siRNA) of the invention.

“Gene silencing” as used herein refers to partial or complete inhibitionof gene expression in a cell and may also be referred to as “geneknockdown.” The extent of gene silencing may be determined by methodsknown in the art, some of which are summarized in InternationalPublication No. WO 99/32619.

As used herein, the terms “ribonucleic acid” and “RNA” refer to amolecule containing at least one ribonucleotide residue. Aribonucleotide is a nucleotide with a hydroxyl group at the 2′ positionof a beta-D-ribo-furanose moiety. These terms include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas modified and altered RNA that differs from naturally occurring RNA bythe addition, deletion, substitution, modification, and/or alteration ofone or more nucleotides. Alterations of an RNA can include addition ofnon-nucleotide material, such as to the end(s) of a siRNA or internally,for example at one or more nucleotides of an RNA.

Nucleotides in an RNA molecule include non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “sense region” is meant a nucleotide sequence of a siRNA moleculehaving complementarity to an antisense region of the siRNA molecule. Inaddition, the sense region of a siRNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siRNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siRNA molecule can include a nucleic acidsequence having complementarity to a sense region of the siRNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. A target nucleic acid can beDNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence either by traditionalWatson-Crick or by other non-traditional modes of binding.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siRNA molecule or the sense andantisense strands of a siRNA molecule. The biodegradable linker isdesigned such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bevariously modulated, for example, by combinations of ribonucleotides,deoxyribonucleotides, and chemically-modified nucleotides, such as2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl,and other 2′-modified or base modified nucleotides. The biodegradablenucleic acid linker molecule can be a dimer, trimer, tetramer or longernucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesin length, or can comprise a single nucleotide with a phosphorus-basedlinkage, for example, a phosphoramidate or phosphodiester linkage. Thebiodegradable nucleic acid linker molecule can also comprise nucleicacid backbone, nucleic acid sugar, or nucleic acid base modifications.

In connection with 2′-modified nucleotides as described herein, by“amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modified orunmodified. Such modified groups are described, for example, inEckstein, et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic, et al.,U.S. Pat. No. 6,248,878.

Supplemental or complementary methods for delivery of nucleic acidmolecules for use within then invention are described, for example, inAkhtar et al., Trends Cell Bio. 2:139, 1992; “Delivery Strategies forAntisense Oligonucleotide Therapeutics,” ed. Akhtar, 1995, Maurer etal., Mol. Membr. Biol. 16:129-140, 1999; Hofland and Huang, Handb. Exp.Pharmacol. 137:165-192, 1999; and Lee et al., ACS Symp. Ser.752:184-192, 2000. Sullivan, et al., International PCT Publication No.WO 94/02595, further describes general methods for delivery of enzymaticnucleic acid molecules.

Nucleic acid molecules can be administered within formulations thatinclude one or more additional components, such as a pharmaceuticallyacceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer,stabilizer, or preservative.

As used herein, the term “carrier” means a pharmaceutically acceptablesolid or liquid diluent, solvent, filler, or encapsulating material.Examples of carriers include saline, biological and pharmaceuticalbuffer systems, and biologically acceptable media. A water-containingliquid carrier can contain pharmaceutically acceptable additives such asacidifying agents, alkalizing agents, antimicrobial preservatives,antioxidants, buffering agents, chelating agents, complexing agents,solubilizing agents, humectants, solvents, suspending and/orviscosity-increasing agents, tonicity agents, wetting agents or otherbiocompatible materials. Examples of ingredients of the above categoriescan be found in the U.S. Pharmacopeia National Formulary, 1990, pp.1857-1859, as well as in Raymond C. Rowe, et al., Handbook ofPharmaceutical Excipients, 5th ed., 2006, and “Remington: The Scienceand Practice of Pharmacy,” 21st ed., 2006, editor David B. Troy.

Examples of preservatives include phenol, methyl paraben, paraben,m-cresol, thiomersal, benzylalkonium chloride, and mixtures thereof.

Examples of surfactants include oleic acid, sorbitan trioleate,polysorbates, lecithin, phosphotidylcholines, various long chaindiglycerides and phospholipids, and mixtures thereof.

Examples of phospholipids include phosphatidylcholine, lecithin,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, andphosphatidylethanolamine, and mixtures thereof.

Examples of dispersants include ethylenediaminetetraacetic acid.

Examples of gases include nitrogen, helium, chlorofluorocarbons (CFCs),hydrofluorocarbons (HFCs), carbon dioxide, air, and mixtures thereof.

In certain embodiments, the siRNA and/or the polypeptide can beencapsulated in liposomes, or reside either internal or external to aliposome, or exist within liposome layers, or be administered byiontophoresis, or incorporated into other vehicles, such as hydrogels,cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, orproteinaceous vectors. See, for example, O'Hare and Normand, PCTInternational Publication No. WO 00/53722. Alternatively, a nucleic acidcomposition can be locally delivered by direct injection or by use of aninfusion pump. Direct injection of the nucleic acid molecules of theinvention, whether subcutaneous, intramuscular, or intradermal, can takeplace using standard needle and syringe methodologies, or by needle-freetechnologies such as those described in Conry et al., Clin. Cancer Res.5:2330-2337, 1999, and Barry et al., International PCT Publication No.WO 99/31262.

The compositions of this invention can be effectively employed aspharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence or severity of, or treat (alleviate one or more symptom(s) toa detectable or measurable extent) of a disease state or other adversecondition in a patient.

In some embodiments, this invention provides pharmaceutical compositionsand methods featuring the presence or administration of one or morepolynucleic acid(s), typically one or more siRNAs, combined, complexed,or conjugated with a lipid, which may further be formulated with apharmaceutically-acceptable carrier, such as a diluent, stabilizer, orbuffer.

Typically, the siRNA will target a gene that is expressed at an elevatedlevel as a causal or contributing factor associated with the subjectdisease state or adverse condition. In this context, the siRNA willeffectively downregulate expression of the gene to levels that prevent,alleviate, or reduce the severity or recurrence of one or moreassociated disease symptoms. Alternatively, for various distinct diseasemodels where expression of the target gene is not necessarily elevatedas a consequence or sequel of disease or other adverse condition, downregulation of the target gene will nonetheless result in a therapeuticresult by lowering gene expression (i.e., to reduce levels of a selectedmRNA and/or protein product of the target gene). Alternatively, siRNAsof the invention may be targeted to lower expression of one gene, whichcan result in upregulation of a “downstream” gene whose expression isnegatively regulated by a product or activity of the target gene.

This siRNAs of this disclosure may be administered in any form, forexample transdermally or by local injection (e.g., local injection atsites of psoriatic plaques to treat psoriasis, or into the joints ofpatients afflicted with psoriatic arthritis or RA). In more detailedembodiments, the invention provides formulations and methods toadminister therapeutically effective amounts of siRNAs directed againstof a mRNA of TNF-α, which effectively down-regulate the TNF-α RNA andthereby reduce or prevent one or more TNF-α-associated inflammatorycondition(s). Comparable methods and compositions are provided thattarget expression of one or more different genes associated with aselected disease condition in animal subjects, including any of a largenumber of genes whose expression is known to be aberrantly increased asa causal or contributing factor associated with the selected diseasecondition.

The compositions of the present invention may also be formulated andused as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions, suspensionsfor injectable administration, and the other forms known in the art.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, for example, systemicadministration, into a cell or patient, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, transepithelial, or by injection. Such formsshould not prevent the composition or formulation from reaching a targetcell (i.e., a cell to which the negatively charged nucleic acid isdesirable for delivery). For example, pharmacological compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.

Examples of agents suitable for formulation with the nucleic acidmolecules of this invention include: P-glycoprotein inhibitors (such asPluronic P85), which can enhance entry of drugs into the CNS(Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol. 13:16-26, 1999);biodegradable polymers, such as poly (DL-lactide-coglycolide)microspheres for sustained release delivery after intracerebralimplantation (Emerich, D. F., et al., Cell Transplant 8:47-58, 1999,Alkermes, Inc., Cambridge, Mass.); and loaded nanoparticles, such asthose made of polybutylcyanoacrylate, which can deliver drugs across theblood brain barrier and can alter neuronal uptake mechanisms (Prog.Neuropsychopharmacol Biol. Psychiatry 23:941-949, 1999). Other examplesof delivery strategies for the nucleic acid molecules of the instantinvention include material described in Boado, et al., J. Pharm. Sci.87:1308-1315, 1998; Tyler, et al., FEBSLett. 421:280-284, 1999;Pardridge, et al., PNAS USA. 92:5592-5596, 1995; Boado, Adv. DrugDelivery Rev. 15:73-107, 1995; Aldrian-Herrada et al., Nucleic AcidsRes. 26:4910-4916, 1998; and Tyler, et al., PNAS USA. 96:7053-7058,1999.

The present invention also includes compositions prepared for storage oradministration, which include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. 1985).For example, preservatives, stabilizers, dyes and flavoring agents maybe provided. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentsmay be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence of, treat, or alleviate a symptom to some extentof a disease state. An amount of from 0.01 mg/kg to 50 mg/kg bodyweight/day of active nucleic acid should be administered.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Additional excipients, for example sweetening,flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension can beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

The siRNAs can also be administered in the form of suppositories, forexample, for rectal administration of the drug. These compositions canbe prepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

The siRNAs can be modified extensively to enhance stability bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H. For a review see Usman andCedergren, TIBS 17:34, 1992; Usman, et al., Nucleic Acids Symp. Ser.31:163, 1994. siRNA constructs can be purified by gel electrophoresisusing general methods or can be purified by high pressure liquidchromatography and re-suspended in water.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency. See for example,Eckstein, et al., International Publication No. WO 92/07065; Perrault etal., Nature 344:565, 1990; Pieken, et al., Science 253, 314, 1991; Usmanand Cedergren, Trends in Biochem. Sci. 17:334, 1992; Usman, et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; and Gold, et al., U.S. Pat. No. 6,300,074. All of the abovereferences describe various chemical modifications that can be made tothe base, phosphate and/or sugar moieties of the nucleic acid moleculesdescribed herein.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications. For areview, see Usman and Cedergren, TIBS 17:34, 1992; Usman, et al.,Nucleic Acids Symp. Ser. 31:163, 1994; Burgin, et al., Biochemistry35:14090, 1996. Sugar modification of nucleic acid molecules have beenextensively described in the art. See Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault, et al. Nature 344:565-568,1990; Pieken, et al. Science 253:314-317, 1991; Usman and Cedergren,Trends in Biochem. Sci. 17:334-339, 1992; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman, et al., J. Biol. Chem. 270:25702, 1995; Beigelman, et al.,International PCT Publication No. WO 97/26270; Beigelman, et al., U.S.Pat. No. 5,716,824; Usman, et al., U.S. Pat. No. 5,627,053; Woolf, etal., International PCT Publication No. WO 98/13526; Thompson, et al.,Karpeisky, et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait,Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein,Annu. Rev. Biochem. 67:99-134, 1998; and Burlina, et al., Bioorg. Med.Chem. 5:1999-2010, 1997. Such publications describe general methods andstrategies to determine the location of incorporation of sugar, baseand/or phosphate modifications and the like into nucleic acid moleculeswithout modulating catalysis. In view of such teachings, similarmodifications can be used as described herein to modify the siRNAnucleic acid molecules of the instant invention so long as the abilityof siRNA to promote RNAi in cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

In some embodiments, the invention features modified siRNA molecules,with phosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 1995, pp. 331-417, and Mesmaeker, et al., “Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research,” ACS, 1994, pp. 24-39.

Methods for the delivery of nucleic acid molecules are described inAkhtar, et al., Trends Cell Bio. 2:139, 1992; “Delivery Strategies forAntisense Oligonucleotide Therapeutics,” ed. Akhtar, 1995; Maurer, etal., Mol. Membr. Biol. 16:129-140, 1999; Hofland and Huang, Handb. Exp.Pharmacol. 137:165-192, 1999; and Lee, et al., ACS Symp. Ser.752:184-192, 2000. Beigelman, et al., U.S. Pat. No. 6,395,713, andSullivan et al., PCT WO 94/02595 further describe the general methodsfor delivery of nucleic acid molecules. These protocols can be utilizedfor the delivery of virtually any nucleic acid molecule. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation internally or externally by liposomes, by iontophoresis,or by incorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see e.g. Gonzalez, et al., Bioconjugate Chem.10:1068-1074, 1999; Wang, et al., International PCT Publication Nos. WO03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac-id (PLGA) and PLCAmicrospheres (see e.g. U.S. Pat. No. 6,447,796 and U.S. PatentApplication Publication No. US 2002130430), biodegradable nanocapsules,and bioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722). Alternatively,the nucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry, et al., Clin. Cancer Res. 5:2330-2337, 1999, and Barry, et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a .beta.-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA orinternally, for example, at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, e.g.Adamic, et al., U.S. Pat. No. 5,998,203, incorporated by referenceherein). These terminal modifications protect the nucleic acid moleculefrom exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety.

Examples of the 3′-cap include, but are not limited to, glyceryl,inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate;3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Lyer, Tetrahedron 49:1925, 1993; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, e.g. Usman and McSwiggen,supra; Eckstein, et al., International PCT Publication No. WO 92/07065;Usman, et al, International PCT Publication No. WO 93/15187; Uhlman &Peyman, supra, all are hereby incorporated by reference herein). Thereare several examples of modified nucleic acid bases known in the art assummarized by Limbach, et al., Nucleic Acids Res. 22:2183, 1994. Some ofthe non-limiting examples of base modifications that can be introducedinto nucleic acid molecules include, inosine, purine, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene,3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines(e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin,et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). By“modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

By “target site” or “target sequence” or “targeted sequence” is meant asequence within a target nucleic acid (e.g., RNA) that is “targeted” forcleavage mediated by a siRNA construct which contains sequences withinits antisense region that are complementary to the target sequence.

The siRNA molecules can be complexed with cationic lipids, packagedwithin liposomes, or otherwise delivered to target cells or tissues. Thenucleic acid or nucleic acid complexes can be locally administered tothrough injection, infusion pump or stent, with or without theirincorporation in biopolymers. In another embodiment, polyethylene glycol(PEG) can be covalently attached to siRNA compounds of the presentinvention, to the polypeptide, or both. The attached PEG can be anymolecular weight, preferably from about 2,000 to about 50,000 daltons(Da).

The sense region can be connected to the antisense region via a linkermolecule, such as a polynucleotide linker or a non-nucleotide linker.

“Inverted repeat” refers to a nucleic acid sequence comprising a senseand an antisense element positioned so that they are able to form adouble stranded siRNA when the repeat is transcribed. The invertedrepeat may optionally include a linker or a heterologous sequence suchas a self-cleaving ribozyme between the two elements of the repeat. Theelements of the inverted repeat have a length sufficient to form adouble stranded RNA. Typically, each element of the inverted repeat isabout 15 to about 100 nucleotides in length, preferably about 20-30 basenucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

“Large double-stranded RNA” refers to any double-stranded RNA having asize greater than about 40 bp for example, larger than 100 bp or moreparticularly larger than 300 bp. The sequence of a large dsRNA mayrepresent a segment of a mRNA or the entire mRNA. The maximum size ofthe large dsRNA is not limited herein. The double-stranded RNA mayinclude modified bases where the modification may be to the phosphatesugar backbone or to the nucleoside. Such modifications may include anitrogen or sulfur heteroatom or any other modification known in theart.

The double-stranded structure may be formed by self-complementary RNAstrand such as occurs for a hairpin or a micro RNA or by annealing oftwo distinct complementary RNA strands.

“Overlapping” refers to when two RNA fragments have sequences whichoverlap by a plurality of nucleotides on one strand, for example, wherethe plurality of nucleotides (nt) numbers as few as 2-5 nucleotides orby 5-10 nucleotides or more.

“One or more dsRNAs” refers to dsRNAs that differ from each other on thebasis of primary sequence.

“Target gene or mRNA” refers to any gene or mRNA of interest. Targetgenes or mRNA may include developmental genes and regulatory genes aswell as metabolic or structural genes or genes encoding enzymes. Thetarget gene may be endogenous or exogenous. The target gene may beexpressed in those cells in which a phenotype is being investigated orin an organism in a manner that directly or indirectly impacts aphenotypic characteristic. Such cells include any cell in the body of anadult or embryonic animal or plant including gamete or any isolated cellsuch as occurs in an immortal cell line or primary cell culture.

Uses for Antisepsis Effects

In some aspects, this disclosure relates generally to the fields ofsepsis. More particularly, this invention relates to compositions andformulations of amino acid lipids and their uses for medicaments and astherapeutics. This invention relates generally to methods of using aminoacid lipids to prevent, treat or ameliorate sepsis, septic shock,inflammatory sepsis, septicemia, or systemic inflammatory responsesyndrome.

Sepsis can be caused by infection when the immune system is compromisedor overwhelmed, or due to certain chemotherapies. For example,endotoxins of gram-negative bacteria can induce septic shock.Gram-negative sepsis is a leading cause of deaths in intensive care.Antimicrobial therapy alone may be insufficient to prevent death insepsis cases. Sepsis can be accompanied by widespread activation of theinnate immune response, leading to uncontrolled production of a varietyof inflammatory mediators. The uncontrolled systemic inflammatoryresponse can cause death.

Amino acid lipids of this disclosure may be active toward reducing theimmune response to certain endotoxins such as lipopolysaccharides.Cationic amphipathic amino acid lipids of this disclosure may bind andneutralize endotoxins, thereby reducing the immune response. Thus, thisdisclosure contemplates uses of amino acid lipids in medicaments andmethods for preventing, treating or ameliorating sepsis, septic shock,inflammatory sepsis, septicemia, or systemic inflammatory responsesyndrome.

Uses for Delivery of Active Agents

The compounds and compositions of this invention may be used fordelivery of any physiologically active agent, as well as any combinationof active agents, as described above or known in the art. The activeagent may be present in the compositions and uses of this invention inan amount sufficient to provide the desired physiological orameliorative effect.

The compounds and compositions of this invention are directed towardenhancing delivery of a range of drug agents and biologically activeagents in mammalian subjects including small molecule compounds anddrugs, peptides, proteins, and vaccine agents.

Examples of active agents include a peptide, a protein, a nucleic acid,a double-stranded RNA, a hematopoietic, an antiinfective; anantidementia; an antiviral, an antitumoral, an antipyretic, ananalgesic, an anti-inflammatory, an antiulcerative, an antiallergenic,an antidepressant, a psychotropic, a cardiotonic, an antiarrythmic, avasodilator, an antihypertensive, a hypotensive diuretic, anantidiabetic, an anticoagulant, a cholesterol-lowering agent, atherapeutic for osteoporosis, a hormone, an antibiotic, a vaccine, acytokine, a hormone, a growth factor, a cardiovascular factor, a celladhesion factor, a central or peripheral nervous system factor, ahumoral electrolyte factor, a hemal organic substance, a bone growthfactor, a gastrointestinal factor, a kidney factor, a connective tissuefactor, a sense organ factor, an immune system factor, a respiratorysystem factor, a genital organ factor, an androgen, an estrogen, aprostaglandin, a somatotropin, a gonadotropin, an interleukin, asteroid, a bacterial toxoid, an antibody, a monoclonal antibody, apolyclonal antibody, a humanized antibody, an antibody fragment, and animmunoglobin.

Examples of active agents include erythropoietin, granulocyte-colonystimulating factor, insulin, Factor VIII, Factor IX, interferon,heparin, hirugen, hirulos, and hirudine.

Examples of active agents include morphine, hydromorphone, oxymorphone,lovorphanol, levallorphan, codeine, nalmefene, nalorphine, nalozone,naltrexone, buprenorphine, butorphanol, or nalbufine, cortisone,hydrocortisone, fludrocortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, dexamethoasone, betamethoasone,paramethosone, fluocinolone, colchicine, acetaminophen, a non-steroidalanti-inflammatory agent NSAID, acyclovir, ribavarin, trifluorothyridine,Ara-A Arabinofuranosyladenine, acylguanosine, nordeoxyguanosine,azidothymidine, dideoxyadenosine, dideoxycytidine, spironolactone,testosterone, estradiol, progestin, gonadotrophin, estrogen,progesterone, papaverine, nitroglycerin, a vasoactive intestinalpeptide, calcitonin gene-related peptide, cyproheptadine, doxepin,imipramine, cimetidine, dextromethorphan, clozaril, superoxidedismutase, neuroenkephalinase, amphotericin B, griseofulvin, miconazole,ketoconazole, tioconazol, itraconazole, fluconazole, cephalosporin,tetracycline, aminoglucoside, erythromicin, gentamicin, polymyxin B,5-fluorouracil, bleomycin, methotrexate, hydroxyurea, dideoxyinosine,floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, I-darubicin,taxol, paclitaxel, tocopherol, quinidine, prazosin, verapamil,nifedipine, diltiazem, tissue plasminogen activator TPA, epidermalgrowth factor EGF, fibroblast growth factor FGF-acidic or basic,platelet derived growth factor PDGF, transforming growth factorTGF-alpha or beta, vasoactive intestinal peptide, tumor necrosis factorTNF, hypothalmic releasing factor, prolactin, thyroid stimulatinghormone TSH, adrenocorticotropic hormone ACTH, parathyroid hormone PTH,follicle stimulating hormone FSF, luteinizing hormone releasing hormoneLHRH, endorphin, glucagon, calcitonin, oxytocin, carbetocin,aldoetecone, enkaphalin, somatostin, somatotropin, somatomedin,alpha-melanocyte stimulating hormone, lidocaine, sufentainil,terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox,buspirone, cromolyn sodium, midazolam, cyclosporin, lisinopril,captopril, delapril, ranitidine, famotidine, superoxide dismutase,asparaginase, arginase, arginine deaminease, adenosine deaminaseribonuclease, trypsin, chemotrypsin, papain, bombesin, substance P,vasopressin, alpha-globulins, transferrin, fibrinogen, beta-lipoprotein,beta-globulin, prothrombin, ceruloplasmin, alpha2-glycoprotein,alpha2-globulin, fetuin, alpha1-lipoprotein, alpha1-globulin, albumin,and prealbumin.

Examples of active agents include opioids or opioid antagonists, such asmorphine, hydromorphone, oxymorphone, lovorphanol, levallorphan,codeine, nalmefene, nalorphine, nalozone, naltrexone, buprenorphine,butorphanol, and nalbufine; corticosterones, such as cortisone,hydrocortisone, fludrocortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, dexamethoasone, betamethoasone,paramethosone, and fluocinolone; other anti-inflammatories, such ascolchicine, ibuprofen, indomethacin, and piroxicam; anti-viral agentssuch as acyclovir, ribavarin, trifluorothyridine, Ara-A(Arabinofuranosyladenine), acylguanosine, nordeoxyguanosine,azidothymidine, dideoxyadenosine, and dideoxycytidine; antiandrogenssuch as spironolactone; androgens, such as testosterone; estrogens, suchas estradiol; progestins; muscle relaxants, such as papaverine;vasodilators, such as nitroglycerin, vasoactive intestinal peptide andcalcitonin related gene peptide; antihistamines, such as cyproheptadine;agents with histamine receptor site blocking activity, such as doxepin,imipramine, and cimetidine; antitussives, such as dextromethorphan;neuroleptics such as clozaril; antiarrhythmics; antiepileptics; enzymes,such as superoxide dismutase and neuroenkephalinase; anti-fungal agents,such as amphotericin B, griseofulvin, miconazole, ketoconazole,tioconazol, itraconazole, and fluconazole; antibacterials, such aspenicillins, cephalosporins, tetracyclines, aminoglucosides,erythromicin, gentamicins, polymyxin B; anti-cancer agents, such as5-fluorouracil, bleomycin, methotrexate, and hydroxyurea,dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin,daunorubicin, I-darubicin, taxol, and paclitaxel; antioxidants, such astocopherols, retinoids, carotenoids, ubiquinones, metal chelators, andphytic acid; antiarrhythmic agents, such as quinidine; antihypertensiveagents such as prazosin, verapamil, nifedipine, and diltiazem;analgesics such as acetaminophen and aspirin; monoclonal and polyclonalantibodies, including humanized antibodies, and antibody fragments;anti-sense oligonucleotides; and RNA, regulatory RNA, interfering RNA,DNA, and viral vectors comprising genes encoding therapeutic peptidesand proteins.

Compositions and Formulations for Administration

As used herein, the terms “administering” and “administration” encompassall means for directly and indirectly delivering a compound orcomposition to a site of action. The compounds and compositions of thisdisclosure may be administered alone, or in combination with othercompounds, compositions, or therapeutic agents which are not disclosedherein.

The compositions and methods of the invention may be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, vaginal, intranasal, intrapulmonary, or transdermaldelivery, or by topical delivery to the eyes, ears, skin or othermucosal surfaces. In some aspects of this invention, the mucosal tissuelayer includes an epithelial cell layer. The epithelial cell can bepulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, orgastrointestinal. Compositions of this invention can be administeredusing conventional actuators such as mechanical spray devices, as wellas pressurized, electrically activated, or other types of actuators.

Compositions of this invention may be administered in an aqueoussolution as a nasal or pulmonary spray and may be dispensed in sprayform by a variety of methods known to those skilled in the art.Pulmonary delivery of a composition of this invention may be achieved byadministering the composition in the form of drops, particles, or spray,which can be, for example, aerosolized, atomized, or nebulized.Pulmonary delivery may be performed by administering the composition inthe form of drops, particles, or spray, via the nasal or bronchialpassages. Particles of the composition, spray, or aerosol can be in aeither liquid or solid form. Preferred systems for dispensing liquids asa nasal spray are disclosed in U.S. Pat. No. 4,511,069. Suchformulations may be conveniently prepared by dissolving compositionsaccording to the present invention in water to produce an aqueoussolution, and rendering said solution sterile. The formulations may bepresented in multi-dose containers, for example in the sealed dispensingsystem disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spraydelivery systems have been described in Transdermal Systemic Medication,Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in U.S. Pat.No. 4,778,810. Additional aerosol delivery forms may include, forexample, compressed air-, jet-, ultrasonic-, and piezoelectricnebulizers, which deliver the biologically active agent dissolved orsuspended in a pharmaceutical solvent, for example, water, ethanol, ormixtures thereof.

Nasal and pulmonary spray solutions of the present invention typicallycomprise the drug or drug to be delivered, optionally formulated with asurface active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent invention, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution may be from about pH 6.8to 7.2. The pharmaceutical solvents employed can also be a slightlyacidic aqueous buffer of pH 4-6. Other components may be added toenhance or maintain chemical stability, including preservatives,surfactants, dispersants, or gases.

In some embodiments, this invention is a pharmaceutical product whichincludes a solution containing a composition of this invention and anactuator for a pulmonary, mucosal, or intranasal spray or aerosol.

A dosage form of the composition of this invention can be liquid, in theform of droplets or an emulsion, or in the form of an aerosol.

A dosage form of the composition of this invention can be solid, whichcan be reconstituted in a liquid prior to administration. The solid canbe administered as a powder. The solid can be in the form of a capsule,tablet or gel.

To formulate compositions for pulmonary delivery within the presentinvention, the biologically active agent can be combined with variouspharmaceutically acceptable additives or delivery-enhancing components,as well as a base or carrier for dispersion of the active agent(s).Examples of additives or delivery-enhancing components include pHcontrol agents such as arginine, sodium hydroxide, glycine, hydrochloricacid, citric acid, and mixtures thereof. Other additives ordelivery-enhancing components include local anesthetics (e.g., benzylalcohol), isotonizing agents (e.g., sodium chloride, mannitol,sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancingagents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g.,serum albumin), and reducing agents (e.g., glutathione). When thecomposition for mucosal delivery is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced in the mucosa at the site of administration. Generally, thetonicity of the solution is adjusted to a value of about ⅓ to 3, moretypically ½ to 2, and most often ¾ to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such aspolyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulosederivatives such as hydroxymethylcellulose, hydroxypropylcellulose,etc., and natural polymers such as chitosan, collagen, sodium alginate,gelatin, hyaluronic acid, and nontoxic metal salts thereof. Abiodegradable polymer may be selected as a base or carrier, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters, etc., can be employed as carriers. Hydrophilic polymers andother carriers can be used alone or in combination, and enhancedstructural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

The biologically active agent can be combined with the base or carrieraccording to a variety of methods, and release of the active agent maybe by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, e.g., isobutyl2-cyanoacrylate (see, e.g., Michael, et al., J. Pharmacy Pharmacol.43:1-5, 1991), and dispersed in a biocompatible dispersing mediumapplied to the nasal mucosa, which yields sustained delivery andbiological activity over a protracted time.

Formulations for mucosal, nasal, or pulmonary delivery may contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10,000 and preferably not more than3000. Examples of hydrophilic low molecular weight compounds includepolyol compounds, such as oligo-, di- and monosaccarides includingsucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose,glycerin, polyethylene glycol, and mixtures thereof. Further examples ofhydrophilic low molecular weight compounds include N-methylpyrrolidone,alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propyleneglycol, etc.), and mixtures thereof.

The compositions of this invention may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and wetting agents, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, andmixtures thereof. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

In certain embodiments of the invention, the biologically active agentmay be administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the invention can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.

Within certain embodiments of this invention, a composition may containone or more natural or synthetic surfactants. Certain naturalsurfactants are found in human lung (pulmonary surfactant), and are acomplex mixture of phospholipids and proteins that form a monolayer atthe alveolar air-liquid interface and reduces surface tension to nearzero at expiration and prevents alveolar collapse. Over 90% (by weight)of pulmonary surfactant is composed of phospholipids with approximately40-80% being DPPC and the remainder being unsaturatedphosphatidylcholines POPG, POPC and phosphatidylglycerols. The remaining10% (by weight) of surfactant is composed of plasma proteins andapoproteins, such as surface proteins (SP)-A, SP-B, SP-C and SP-D.

Examples of natural surfactants that may be used in this inventioninclude SURVANTA™ (beractant), CUROSURF™ (poractant alfa) and INFASURF™(calfactant), and mixtures thereof.

Examples of synthetic surfactants include sinapultide; a combination ofdipalmitoylphosphatidylcholine, palmitoyloleoyl phosphatidylglycerol andpalmitic acid; SURFAXIN™ (lucinactant); and EXOSURF™ (colfosceril);components which may contain tyloxapol, DPPC, and hexadecanol; andmixtures thereof.

Compositions of this invention can be prepared by methods known in theart. Methods of making the lipid compositions include ethanol injectionmethods and extrusion methods using a Northern Lipids Lipex Extrudersystem with stacked polycarbonate membrane filters of defined pore size.Sonication using probe tip and bath sonicators can be employed toproduce lipid particles of uniform size. Homogenous and monodisperseparticle sizes can be obtained without the addition of the nucleic acidcomponent. For in vitro transfection compositions, the nucleic acidcomponent can be added after the transfection agent is made andstabilized by additional buffer components. For in vivo deliverycompositions, the nucleic acid component is part of the formulation.

The compositions and formulations of this invention may be administeredby various routes, for example, to effect systemic delivery viaintravenous, parenteral, or intraperitoneal routes. In some embodiments,an agent may be delivered intracellularly, for example, in cells of atarget tissue such as lung or liver, or in inflamed tissues. Includedwithin this disclosure are compositions and methods for delivery of anagent by removing cells of a subject, delivering an agent to the removedcells, and reintroducing the cells into a subject. In some embodiments,this invention provides a method for delivery of an agent in vivo. Acomposition may be administered intravenously, subcutaneously, orintraperitoneally to a subject. In some embodiments, the inventionprovides methods for in vivo delivery of an agent to the lung of amammalian subject.

ADDITIONAL EMBODIMENTS

All publications, references, patents, patent publications and patentapplications cited herein are each hereby specifically incorporated byreference in entirety.

While this invention has been described in relation to certainembodiments, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that thisinvention includes additional embodiments, and that some of the detailsdescribed herein may be varied considerably without departing from thisinvention. This invention includes such additional embodiments,modifications and equivalents. In particular, this invention includesany combination of the features, terms, or elements of the variousillustrative components and examples.

The use herein of the terms “a,” “an,” “the” and similar terms indescribing the invention, and in the claims, are to be construed toinclude both the singular and the plural.

The terms “comprising,” “having,” “including” and “containing” are to beconstrued as open-ended terms which mean, for example, “including, butnot limited to.” Thus, terms such as “comprising,” “having,” “including”and “containing” are to be construed as being inclusive, not exclusive.

Recitation of a range of values herein refers individually to each andany separate value falling within the range as if it were individuallyrecited herein, whether or not some of the values within the range areexpressly recited. For example, the range “4 to 12” includes withoutlimitation the values 5, 5.1, 5.35 and any other whole, integer,fractional, or rational value greater than or equal to 4 and less thanor equal to 12. Specific values employed herein will be understood asexemplary and not to limit the scope of the invention.

Recitation of a range of number of carbon atoms herein refersindividually to each and any separate value falling within the range asif it were individually recited herein, whether or not some of thevalues within the range are expressly recited. For example, the term“C1-22” includes without limitation the species C1, C2, C3, C4, C5, C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21,and C22.

Definitions of technical terms provided herein should be construed toinclude without recitation those meanings associated with these termsknown to those skilled in the art, and are not intended to limit thescope of the invention. Definitions of technical terms provided hereinshall be construed to dominate over alternative definitions in the artor definitions which become incorporated herein by reference to theextent that the alternative definitions conflict with the definitionprovided herein.

The examples given herein, and the exemplary language used herein aresolely for the purpose of illustration, and are not intended to limitthe scope of the invention.

When a list of examples is given, such as a list of compounds ormolecules suitable for this invention, it will be apparent to thoseskilled in the art that mixtures of the listed compounds or moleculesare also suitable.

EXAMPLES Example 1 Preparation of C10-Arg-C10N-(5-guanidino-1-oxo-1-(decylamino)pentan-2-yl)decanamide

Fmoc-Arg(Pbf)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 5.06 g (11.7 mmol, 3 eq)of Fmoc-Arg(Pbf)-OH (Mw=648.8, Novabiochem, 04-12-1145) and 2.23 ml(12.87 mmol, 3.3 eq) of DIPEA (Aldrich, Mw=129.2, d=0.74) were added.

Arg(Pbf)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 30 mlof 20% piperidine/DMF for 30 min.

C10-Arg(Pbf)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of decanoic acid (Sigma, Mw=127.27), 5.54 g (11.7 mmol) of HCTU(Mw=473.7) and 2.23 ml (12.87 mmol) of DIPEA (Mw=129.2, d=0.74) in 30 mlof DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C10-Arg(Pbf)-OH (Mw=580.83) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C10-Arg(Pbf)-C10 (Mw=720.13). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C10-amine (Sigma, Mw=157.3, d=0.792), 5.54 g (11.7 mmol) ofHCTU (Mw=473.7) and 2.23 ml (12.87 mmol) of DIPEA (Mw=129.2, d=0.74) in50 ml of DMF were added. After 1 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO3 and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C10-Arg-C10 (Mw=467.8) To the oily residue from the previous reaction100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrs solvent wasevaporated. Residue was dissolved in AcCN/0.5 M HCl 1:1 and purified byRP_Akta Explorer on C18 Phenomenex column (Phenomenex RP, 250×21.2 mm,Serial No.: 234236-1, Column volume 83 ml) and eluted with 0-100%acetonitrile gradient using water as mobile phase within 3 CV; X=215 nm.Acetonitrile was evaporated and the product was lyophilized.

Example 2 Preparation of C10-D-Arg-C10N-(5-guanidino-1-oxo-1-(decylamino)pentan-2-yl)decanamide

Fmoc-D-Arg(Pmc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 5.06 g (11.7 mmol, 3 eq)of Fmoc-D-Arg(Pmc)-OH (M_(w)=662.8, Novabiochem, 04-12-1145) and 2.23 ml(12.87 mmol, 3.3 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

D-Arg(Pmc)-resin. After 2 hrs the resin was washed 3× withDCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group wasdeprotected with 30 ml of 20% piperidine/DMF for 30 min.

C10-D-Arg(Pmc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of decanoic acid (Sigma, M_(w)=127.27), 5.54 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C10-D-Arg(Pmc)-OH (M_(w)=591.84) was cleaved from the resin by 1%TFA/DCM (5×30 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C10-D-Arg(Pmc)-C10 (M_(w)=732.14). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C10-amine (Sigma, M_(w)=157.3, d=0.792), 5.54 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 1 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C10-D-Arg-C10 (M_(w)=467.8) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Residue was dissolved in AcCN/0.5 M HCl 1:1 andpurified by RP_Akta Explorer on C18 Phenomenex column (Phenomenex RP,250×21.2 mm, Serial No.: 234236-1, Column volume 83 ml) and eluted with0-100% acetonitrile gradient using water as mobile phase within 3 CV;λ=215 nm. Acetonitrile was evaporated and the product was lyophilized.

Example 3 Preparation of C12-Arg-C12N-(5-guanidino-1-oxo-1-(dodecylamino)pentan-2-yl)dodecanamide

Fmoc-Arg(Pbf)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 200ml reaction vessel for solid phase synthesis, 8.434 g (13 mmol, 2 eq) ofFmoc-Arg(Pbf)-OH (M_(w)=648.8, Novabiochem, 04-12-1145) and 2.26 ml (13mmol, 2 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Arg(Pbf)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected 2× with 50ml of 20% piperidine/DMF for 15 min.

C12-Arg(Pbf)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.95 g (9.75 mmol)of dodecanoic acid (Sigma, M_(w)=200.32), 4.033 (9.75 mmol) of HCTU(M_(w)=417.7) and 1.7 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C12-Arg(Pbf)-OH (M_(w)=608.9) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C12-Arg(Pbf)-C12 (M_(w)=776.13). Second coupling was carried out insolution. To the oily residue from the previous step 1.246 g (6.5 mmol)of EDC*HCl (M_(w)=191.7), 1.06 g (7.15 mmol, 1.1 eq) of HOBt*H₂O(M_(w)=153), and 5.65 ml (31.5 mmol, 5 eq) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DCM were added to preactivate —COOH followed by 1.32g (7.15 mmol) of C12-amine (Sigma, M_(w)=185.36) 20 min later. After 1hr 100 ml of AcOEt was added and organic layer was washed in separatoryfunnel with 3×0.5 M HCl, 3×10% NaCO₃ and 3×NaCl. AcOEt layer was driedwith anhydrous MgSO₄ and evaporated. The product was purified onTELEDYNE Isco CombiFlash R_(f) instrument, 40 g normal phase silica gelcolumn, 100% DCM for 5 CV(column volume) and 0-5% MeOH for 10 CV,detection 254 nm, flow 40 ml/min.

C12-Arg-C12 (M_(w)=523.8) To the oily residue from the previous reaction50 ml of 85% TFA/DCM 2.5% TIS was added and after 3 hrs solvent wasevaporated. The product was precipitated with 0.5 M HCl.

Example 4 Preparation of C12-Arg-C14N-(5-guanidino-1-oxo-1-(tetradecylamino)pentan-2-yl)dodecanamide

Fmoc-Arg(Pbf)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 200ml reaction vessel for solid phase synthesis, 8.434 g (13 mmol, 2 eq) ofFmoc-Arg(Pbf)-OH (M_(w)=648.8, Novabiochem, 04-12-1145) and 2.26 ml (13mmol, 2 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Arg(Pbf)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected 2* with 50ml of 20% piperidine/DMF for 15 min.

C12-Arg(Pbf)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.95 g (9.75 mmol)of dodecanoic acid (Sigma, M_(w)=200.32), 4.033 (9.75 mmol) of HCTU(M_(w)=417.7) and 1.7 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C12-Arg(Pbf)-OH (M_(w)=608.9) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C12-Arg(Pbf)-C14 (M_(w)=804.13). Second coupling was carried out insolution. To the oily residue from the previous step 1.246 g (6.5 mmol)of EDC*HCl (M_(w)=191.7), 1.06 g (7.15 mmol, 1.1 eq) of HOBt*H₂O(M_(w)=153), and 5.65 ml (31.5 mmol, 5 eq) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DCM were added to preactivate —COOH followed by 1.52g (7.15 mmol) of C14-amine (Sigma, M_(w)=213.41) 20 min later. After 1hr 100 ml of AcOEt was added and organic layer was washed in separatoryfunnel with 3×0.5 M HCl, 3×10% NaCO₃ and 3×NaCl. AcOEt layer was driedwith anhydrous MgSO₄ and evaporated. The product was purified onTELEDYNE Isco CombiFlash R_(f) instrument, 40 g normal phase silica gelcolumn, 100% DCM for 5 CV(column volume) and 0-5% MeOH for 10 CV,detection 254 nm, flow 40 ml/min.

C12-Arg-C14 (M_(w)=551.8) To the oily residue from the previous reaction50 ml of 85% TFA/DCM 2.5% TIS was added and after 3 hrs solvent wasevaporated. The product was precipitated with 0.5 M HCl.

Example 5

In like fashion to Examples 1-4 were made C14-Arg-C14 (Yield: 1.6 g),C16-Arg-C16, and C18-Arg-C18.

Example 6 Preparation of C18(oleic)-Arg-C16N-(5-guanidino-1-oxo-1-(hexadecylamino)pentan-2-yl)octadec-9-enamide

Fmoc-Arg(Pbf)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 60ml reaction vessel for solid phase synthesis, 8.4 g (13 mmol, 2 eq) ofFmoc-Arg(Pbf)-OH (M_(w)=648.8, Novabiochem, 04-12-1145) and 2.26 ml (13mmol, 2 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Arg(Pbf)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected 2 timeswith 50 ml of 20% piperidine/DMF for 15 min.

C18_(oleic)-Arg(Pbf)-resin. After Fmoc deprotection the resin was washedwith 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.1 ml (9.75mmol) of oleic acid (Sigma, M_(w)=282.47; d=0.891), 4 g (9.75 mmol) ofHCTU (M_(w)=413.7) and 1.7 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74)in 50 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18_(oleic)-Arg(Pbf)-OH (M_(w)=690.83) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18_(oleic)-Arg(Pbf)-C16 (M_(w)=850.13). Second coupling was carried outin solution. To the oily residue from the previous step 1.437 g (7.5mmol) of EDC*HCl (M_(w)=191.7), 1.178 g (7.7 mmol,) of HOBt*H₂O(M_(w)=153), and 6.5 ml (37.5 mmol, 5 eq) of DIPEA (M_(w)=129.2, d=0.74)in 50 ml of DCM were added to preactivate —COOH followed by addition of1.86 g (7.7 mmol) of C16-amine (Sigma, M_(w)=241.46) 20 min later. Afterovernight reaction organic layer was washed in separatory funnel with3×0.5 M HCl, 3×10% NaCO₃ and 3×NaCl. DCM layer was dried with anhydrousMgSO₄ and evaporated. The product was purified on TELEDYNE IscoCombiFlash R_(f) instrument, 40 g normal phase silica gel column, 100%DCM for 5 CV(column volume) and 0-5% MeOH for 10 CV, detection 215 nm,flow 40 ml/min.

C18_(oleic)-Arg-C16 (M_(w)=661.8) To the oily residue from the previousreaction, 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Product was precipitated with 0.5 M HCl andrepurified. Yield: 1.8 g.

Example 7 Preparation of C8-homoArg-C8N-(6-guanidino-1-oxo-1-(octylamino)hexan-2-yl)octanamide

Fmoc-hArg(diBoc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 3.57 g (5.85 mmol, 1.5 eq)of Fmoc-hArg(diBoc)-OH (M_(w)=610.69, Novabiochem,) and 2.04 ml (11.7mmol, 3.0 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

hArg(diBoc)-resin. After 2 hrs the resin was washed 3× withDCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group wasdeprotected with 30 ml of 20% piperidine/DMF for 30 min.

C8-hArg(diBoc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.854 ml (11.7mmol) of C8 acid (Sigma, M_(w)=144.22, d=0.91), 4.84 g (11.7 mmol) ofHCTU (M_(w)=413.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C8-hArg(diBoc)-OH (M_(w)=514.68) was cleaved from the resin by 1%TFA/DCM (5×30 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C8-hArg(diBoc)-C8 (M_(w)=625.93). Second coupling was carried out insolution. To the oily residue from the previous reaction 1.93 ml (11.7mmol) of C8-amine (Sigma, M_(w)=129.25, d=0.782), 4.84 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 1 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C8-hArg-C8 (M_(w)=426.8) To the oily residue from the previous reaction100 ml of 95% TFA/DCM 2.5% TIS was added and after 1 hr solvent wasevaporated. Residue was dissolved in MeOH/AcCN/0.5 M HCl 1:1:1 andpurified by RP_Akta Explorer on C18 Phenomenex column (Phenomenex RP,250×21.2 mm, Serial No.: 234236-1, Column volume 83 ml) and eluted with50-100% acetonitrile gradient using water as mobile phase within 3 CV;λ=215 nm. Acetonitrile was evaporated and the product was lyophilized.

Example 8 Preparation of C10-homoArg-C10N-(6-guanidino-1-oxo-1-(decylamino)hexan-2-yl)decanamide

Fmoc-hArg(diBoc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 3.57 g (5.85 mmol, 1.5 eq)of Fmoc-hArg(diBoc)-OH (M_(w)=610.69, Novabiochem,) and 2.04 ml (11.7mmol, 3.0 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

hArg(diBoc)-resin. After 2 hrs the resin was washed 3× withDCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group wasdeprotected with 30 ml of 20% piperidine/DMF for 30 min.

C10-hArg(diBoc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of decanoic acid (Sigma, M_(w)=127.27), 5.54 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C10-hArg(diBoc)-OH (M_(w)=541.78) was cleaved from the resin by 1%TFA/DCM (5×30 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C10-hArg(diBoc)-C10 (M_(w)=682.08). Second coupling was carried out insolution.

To the oily residue from the previous reaction 2.32 ml (11.7 mmol) ofC10-amine (Sigma, M_(w)=157.3, d=0.792), 5.54 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 1 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C10-hArg-C10 (M_(w)=481.8) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 1 hr solventwas evaporated. Residue was dissolved in MeOH/0.5 M HCl 1:1 andprecipitated with H₂O.

Example 9 Preparation of C12-homoArg-C12N-(6-guanidino-1-oxo-1-(dodecylamino)hexan-2-yl)dodecanamide

In like fashion to Example 8 was made C12-homoArg-C12 (Yield: 1 g).

Example 10 Preparation of C8-nor-norArg-C8N-(3-guanidino-1-oxo-1-(octylamino)propan-2-yl)octanamide

Fmoc-Dap(Boc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.5 g (5.85 mmol, 1.5 eq)of Fmoc-Dap(Boc)-OH (M_(w)=426.5, (Fmoc-(N-β-Boc)-L-α,β-diaminopropionicacid), AnaSpec, 22140) and 2.03 ml (11.7 mmol, 3.0 eq) of DIPEA(Aldrich, M_(w)=129.2, d=0.74) were added.

Dap-(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 30 mlof 20% piperidine/DMF for 30 min.

C8-Dap(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.236 ml (11.7mmol) of C8 acid (Sigma, M_(w)=144.22, d=0.910), 4.84 g (11.7 mmol) ofHCTU (M_(w)=413.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C8-Dap(Boc)-OH (M_(w)=330.49) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C8-Dap(Boc)-C8 (M_(w)=441.74). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C8-amine (Sigma, M_(w)=129.25, d=0.782), 4.84 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 1 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C8-Dap-C8 (M_(w)=341.74). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C8-nor-norArg(diBoc)-C8 (M_(w)=583.74). The residue was dissolved in 50ml of DCM and pH was adjusted to 9 with TEA. 2.23 g (5.85 mM, 1.5 eq) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 4 hrs DCM was evaporated.

C8-nor-norArg-C8 (M_(w)=384.8) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Residue was dissolved in MeOH/AcCN/0.5 M HCl1:1:1 and purified by RP_Akta Explorer on C18 Phenomenex column(Phenomenex RP, 250×21.2 mm, Serial No.: 234236-1, Column volume 83 ml)and eluted with 50-100% acetonitrile gradient using water as mobilephase within 3 CV; λ=215 nm. Acetonitrile was evaporated and the productwas lyophilized.

Example 11 Preparation of C10-nor-norArg-C10N-(3-guanidino-1-oxo-1-(decylamino)propan-2-yl)decanamide

Fmoc-Dap(Boc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.5 g (5.85 mmol, 1.5 eq)of Fmoc-)Dap(Boc)-OH (M_(w)=426.5,(Fmoc-(N-β-Boc)-L-α,β-diaminopropionic acid), AnaSpec, 22140) and 2.03ml (11.7 mmol, 3.0 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) wereadded.

Dap(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 30 mlof 20% piperidine/DMF for 30 min.

C10-Dap(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of decanoic acid (Sigma, M_(w)=172.27), 4.83 g (11.7 mmol) of HCTU(M_(w)=413.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C10-Dap(Boc)-OH (M_(w)=357.54) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C10-Dap(Boc)-C10 (M_(w)=496.84). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C10-amine (Sigma, M_(w)=157.3, d=0.792), 4.83 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 2 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C10-Dap-C10 (M_(w)=397.8). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C10-nor-norArg(diBoc)-C10 (M_(w)=640.8). The residue was dissolved in 50ml of DCM and pH was adjusted to 9 with TEA. 2.23 g (5.85 mM, 1.5 eq) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 4 hrs DCM was evaporated.

C10-nor-norArg-C10 (M_(w)=439.8) To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated. Residue was dissolved in AcCN/MeOH/0.5 M HCl1:1:1 and purified by RP_Akta Explorer on C18 Phenomenex column(Phenomenex RP, 250×21.2 mm, Serial No.: 234236-1, Column volume 83 ml)and eluted with 50-100% acetonitrile gradient using water as mobilephase within 3 CV; λ=215 nm. Acetonitrile was evaporated and the productwas lyophilized. Yield: 1.6 g.

Example 12 Preparation of C12-nor-norArg-C12N-(3-guanidino-1-oxo-1-(dodecylamino)propan-2-yl)dodecanamide

Fmoc-Dap(Boc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.5 g (5.85 mmol, 1.5 eq)of Fmoc-Dap(Boc)-OH (M_(w)=426.5, (Fmoc-(N-3-Boc)-L-α,β-diaminopropionicacid), AnaSpec, 22140) and 2.03 ml (11.7 mmol, 3.0 eq) of DIPEA(Aldrich, M_(w)=129.2, d=0.74) were added.

Dap(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 30 mlof 20% piperidine/DMF for 30 min.

C12-Dap(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 2.34 g (11.7 mmol)of C12-acid (Sigma, M_(w)=200.32), 4.48 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C12-Dap(Boc)-OH (M_(w)=386.59) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C12-Dap(Boc)-C12 (M_(w)=553.95). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.168 g (11.7mmol) of C12-amine (Sigma, M_(w)=185.36), 4.48 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 1 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C12-Dap-C12 (M_(w)=453.9). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C12-nor-norArg(diBoc)-C12 (M_(w)=696.13). The residue was dissolved in50 ml of DCM and pH was adjusted to 9 with TEA. 2.23 g (5.85 mM, 1.5 eq)of 1,3-Di-Boc-2-(trifluoromethylsulfonyl) guanidine (M_(w)=391.36,Aldrich 15033) was added and after 4 hrs DCM was evaporated.

Example 13 Preparation of C8-norArg-C8N-(4-guanidino-1-oxo-1-(octylamino)butan-2-yl)octanamide

Fmoc-Dab(Boc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.577 g (5.85 mmol, 1.5eq) of Fmoc-Dab(diBoc)-OH (M_(w)=440.5,(Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyric acid, AnaSpec, 28246) and 2.23 ml(12.87 mmol, 3.3 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Arg-Dab(Boc)-resin. After 2 hrs the resin was washed 3× withDCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group wasdeprotected with 30 ml of 20% piperidine/DMF for 30 min.

C8-Dab(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of C8 acid (Sigma, M_(w)=144.22, d=0.910), 4.84 g (11.7 mmol) of HCTU(M_(w)=413.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C8-Dab(Boc)-OH (M_(w)=344.49) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C8-Dab(Boc)-C8 (M_(w)=455.74). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C8-amine (Sigma, M_(w)=129325, d=0.782), 4.84 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 1 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C8-Dab-C8 (M_(w)=355.74). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C8-norArg(diBoc)-C8 (M_(w)=597.74). The residue was dissolved in 50 mlof DCM and pH was adjusted to 9 with TEA. 2.23 g (5.85 mM, 1.5 eq) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 4 hrs DCM was evaporated.

C8-norArg-C8 (M_(w)=398.8) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Residue was dissolved in MeOH/AcCN/0.5 M HCl1:1:1 and purified by RP_Akta Explorer on C18 Phenomenex column(Phenomenex RP, 250×21.2 mm, Serial No.: 234236-1, Column volume 83 ml)and eluted with 50-100% acetonitrile gradient using water as mobilephase within 3 CV; λ=215 nm. Acetonitrile was evaporated and the productwas lyophilized. Yield: 1.1 g.

Example 14

In like fashion to Example 13 were made C10-norArg-C10 (Yield: 1.2 g),C12-norArg-C12 (Yield: 2.9 g), C14-norArg-C14 (Yield: 630 mg), andC16-norArg-C16 (Yield: 1.0 g).

Example 15 Preparation of C12-norArg-C12N-(4-guanidino-1-oxo-1-(dodecylamino)butan-2-yl)dodecanamide

Fmoc-Dab(Boc)-resin. To 8 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 100 ml of dry DCM in500 ml reaction vessel for solid phase synthesis, 5 g (11.35 mmol, 1.2eq) of Fmoc-Dab(Boc)-OH (M_(w)=440.5,(Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyric acid, AnaSpec, 28246) and 4 ml(22.7 mmol, 2.0 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 2 hrs of shaking on the shaker the resin waswashed 3× with DCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmocgroup was deprotected 2 times with 50 ml of 20% piperidine/DMF for 15min.

C12-Dab(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 4.54 g (22.7 mmol)of C12-acid (Sigma, M_(w)=200.32), 9.48 g (22.7 mmol) of HCTU(M_(w)=413.7) and 4.52 ml (26 mmol) of DIPEA (M_(w)=129.2, d=0.74) in100 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C12-Dab(Boc)-OH (M_(w)=400.59) was cleaved from the resin by 1% TFA/DCM(5×50 ml for 2 min was filtered to flask with 10 ml 10% pyridine/MeOH)and solvent was evaporated.

C12-Dab(Boc)-C12 (M_(w)=567.95). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.168 g (11.7mmol) of C12-amine (Sigma, M_(w)=185.36), 4.48 g (11.7 mmol) of HCTU(M_(w)=413.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 1 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.1 M HCl, 3×5%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C12-Dab-C12 (M_(w)=467.9). To the oily residue from the previousreaction, 150 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C12-norArg(diBoc)-C12 (M_(w)=710.13). The residue was dissolved in 50 mlof DCM and pH was adjusted to 9-10 with TEA. 9 g (23 mM) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 3 hrs DCM was evaporated. The product waspurified on TELEDYNE Isco CombiFlash R_(f) instrument, 120 g normalphase silica gel column, 100% DCM for 5.4 CV(column volume) and 0-5%MeOH for 6.3 CV, detection 215 nm, flow 70 ml/min.

C12-norArg-C12 (M_(w)=509.92) To the residue from the previous reaction150 ml of 70% TFA/DCM 2.5% TIS was added and after 1 hr solvent wasevaporated. Residue was precipitated by 0.5 M HCl.

Example 16 Preparation of C18-norArg-C18N-(4-guanidino-1-oxo-1-(octadecylamino)butan-2-yl)octadecanamide

Fmoc-Dab(Boc)-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.577 g (5.85 mmol, 1.5eq) of Fmoc-Dab(Boc)-OH (M_(w)=440.5,(Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyric acid, AnaSpec, 28246) and 2.03 ml(11.7 mmol, 3.0 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 30 mlof 20% piperidine/DMF for 30 min.

C18-Dab(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM2 and for the coupling reaction 2.21 g (7.8 mmol)of C18 acid (Sigma, M_(w)=284.48), 3.32 g (7.8 mmol) of HCTU(M_(w)=473.7) and 1.49 ml (8.58 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18-Dab(Boc)-OH (M_(w)=568.56) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C18-Dab(Boc)-C18 (M_(w)=735.95). Second coupling was carried out insolution. To the oily residue from the previous reaction, 2.1 g (7.8mmol) of C18-amine (Sigma, M_(w)=269.52), 3.32 g (7.8 mmol) of HCTU(M_(w)=473.7) and 1.49 ml (8.58 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 1 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C18-Dab-C18 (M_(w)=635.9). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 1 hrsolvent was evaporated.

C18-norArg(diBoc)-C18 (M_(w)=872.13). The residue was dissolved in 50 mlof DCM and pH was adjusted to 9 with TEA. 2.23 g (5.85 mM, 1.5 eq) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 4 hrs DCM was evaporated.

C18-norArg-C18 (M_(w)=677.92) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Residue was precipitated by mixture of AcCN/0.5M HCl 1:1.

Example 17 Preparation of C18(oleic)-norArg-C8N-(4-guanidino-1-oxo-1-(octylamino)butan-2-yl)octadec-9-enamide

Fmoc-Dab(Boc)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 50ml reaction vessel for solid phase synthesis, 5.726 g (13 mmol, 2 eq) ofFmoc-Dab(Boc)-OH (M_(w)=440.5, (Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyricacid, AnaSpec, 28246) and 2.26 ml (13 mmol, 2.0 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 40 mlof 20% piperidine/DMF 2 times for 30 min.

C18oleic-Dab(Boc)-resin. After Fmoc deprotection the resin was washedwith 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g (13mmol) of oleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol) ofHCTU (M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2, d=0.74)in 50 ml of DMF were added.

After 2 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18oleic-Dab(Boc)-OH (M_(w)=538.56) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18oleic-Dab(Boc)-C8 (M_(w)=594.01). Second coupling was carried out insolution. To the oily residue from the previous reaction, 1.227 g (9.75mmol) of C8-amine (Sigma, M_(w)=129.25), 4.033 g (9.75 mmol) of HCTU(M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 4 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C18oleic-Dab-C8 (M_(w)=493.92). To the oily residue from the previousreaction, 70 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was removed under reduced pressure.

C18oleic-norArg(diBoc)-C8 (M_(w)=735.95). The residue was dissolved in50 ml of DCM and pH was adjusted to 9 with TEA. 2.543 g (6.5 mM, 1 eq)of 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36,Aldrich 15033) was added and after 4 hrs DCM was evaporated.

C18oleic-norArg-C8 (M_(w)=535.89) To the oily residue from the previousreaction 100 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was evaporated. Crude product was purified on TELEDYNE IscoCombiFlash R_(f) instrument using 40 g normal phase silica gel column,100% DCM for 3 CV(column volume) and 0-15% MeOH for 7 CV, detection 214nm, flow 45 ml/min TLC: Rf=0.2 (CH₂Cl₂:MeOH=9:1). DCM/MeOH wasevaporated and residue was precipitated by 0.1M HCl. Yield: 0.7 g.

Example 18 Preparation of C18(oleic)-norArg-C12N-(4-guanidino-1-oxo-1-(dodecylamino)butan-2-yl)octadec-9-enamide

Fmoc-Dab(Boc)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 50ml reaction vessel for solid phase synthesis, 5.726 g (13 mmol, 2 eq) ofFmoc-Dab(Boc)-OH (M_(w)=440.5, (Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyricacid, AnaSpec, 28246) and 2.26 ml (13 mmol, 2.0 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 40 mlof 20% piperidine/DMF 2 times for 30 min.

C18oleic-Dab(Boc)-resin. After Fmoc deprotection the resin was washedwith 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g (13mmol) of oleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol) ofHCTU (M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2, d=0.74)in 50 ml of DMF were added.

After 2 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18oleic-Dab(Boc)-OH (M_(w)=538.56) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18oleic-Dab(Boc)-C12 (M_(w)=650.03). Second coupling was carried out insolution. To the oily residue from the previous reaction, 1.76 g (9.75mmol) of C12-amine (Sigma, M_(w)=185.36), 4.033 g (9.75 mmol) of HCTU(M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 4 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C18oleic-Dab-C12 (M_(w)=549.91). To the oily residue from the previousreaction, 70 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was removed under reduced pressure.

C18oleic-norArg(diBoc)-C12 (M_(w)=792.5). The residue was dissolved in50 ml of DCM and pH was adjusted to 9 with TEA. 2.543 g (6.5 mM, 1 eq)of 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36,Aldrich 15033) was added and after 4 hrs DCM was evaporated.

C18oleic-norArg-C12 (M_(w)=591.55) To the oily residue from the previousreaction 100 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was evaporated. Crude product was purified on TELEDYNE IscoCombiFlash R_(f) instrument using 40 g normal phase silica gel column,100% DCM for 3 CV(column volume) and 0-20% MeOH for 10 CV, detection 214nm, flow 45 ml/min. TLC: Rf=0.2 (CH₂Cl₂:MeOH=9:1) DCM/MeOH wereevaporated and residue was precipitated by 0.1M HCl.

Example 19 Preparation of C18(oleic)-norArg-C16N-(4-guanidino-1-oxo-1-(hexadecylamino)butan-2-yl)octadec-9-enamide

Fmoc-Dab(Boc)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 50ml reaction vessel for solid phase synthesis, 5.726 g (13 mmol, 2 eq) ofFmoc-Dab(Boc)-OH (M_(w)=440.5, (Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyricacid, AnaSpec, 28246) and 2.26 ml (13 mmol, 2.0 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 40 mlof 20% piperidine/DMF 2 times for 30 min.

C18oleic-Dab(Boc)-resin. After Fmoc deprotection the resin was washedwith 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g (13mmol) of oleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol) ofHCTU (M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2, d=0.74)in 50 ml of DMF were added.

After 2 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18oleic-Dab(Boc)-OH (M_(w)=538.56) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18oleic-Dab(Boc)-C16 (M_(w)=705.95). Second coupling was carried out insolution. To the oily residue from the previous reaction, 2.354 g (9.75mmol) of C16-amine (Sigma, M_(w)=241.46), 4.033 g (9.75 mmol) of HCTU(M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 4 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C18oleic-Dab-C16 (M_(w)=605.9). To the oily residue from the previousreaction, 70 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was removed under reduced pressure.

C18oleic-norArg(diBoc)-C16 (M_(w)=842.13). The residue was dissolved in50 ml of DCM and pH was adjusted to 9 with TEA. 2.543 g (6.5 mM, 1 eq)of 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36,Aldrich 15033) was added and after 4 hrs DCM was evaporated.

C18oleic-norArg-C16 (M_(w)=647.92) To the oily residue from the previousreaction 100 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was evaporated. Crude product was purified on TELEDYNE IscoCombiFlash R_(f) instrument using 48 g normal phase silica gel column,100% DCM for 3 CV(column volume) and 0-20% MeOH for 10 CV, detection 214nm, flow 45 ml/min. DCM/MeOH was evaporated and residue was precipitatedby 0.1M HCl.

Example 20 Preparation of C18(oleic)-norArg-C18N-(4-guanidino-1-oxo-1-(octadecylamino)butan-2-yl)octadec-9-enamide

Fmoc-Dab(Boc)-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 60ml reaction vessel for solid phase synthesis, 5.726 g (13 mmol, 2 eq) ofFmoc-Dab(Boc)-OH (M_(w)=440.5, (Fmoc-(N-γ-Boc)-L-α,γ-diaminobutyricacid, AnaSpec, 28246) and 2.26 ml (13 mmol, 2.0 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added.

Dab(Boc)-resin. After 3 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 50 mlof 20% piperidine/DMF twice for 15 min.

C18:1-Dab(Boc)-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g (13 mmol) ofoleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol) of HCTU(M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2, d=0.74) in 50ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18:1-Dab(Boc)-OH (M_(w)=566.56) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18:1-Dab(Boc)-C18:1 (M_(w)=734.95). Second coupling was carried out insolution. To the oily residue from the previous reaction, 3.725 g (9.75mmol) of oleyl amine (Sigma, M_(w)=267.49, 70%), 4.033 g (9.75 mmol) ofHCTU (M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 4 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated.

C18:1-Dab-C18 (M_(w)=635.9). To the oily residue from the previousreaction, 100 ml of 80% TFA/DCM 2.5% TIS was added and after 20 minsolvent was evaporated.

C18:1-norArg(diBoc)-C18:1 (M_(w)=874.13). The residue was dissolved in50 ml of DCM and pH was adjusted to 9 with TEA. 2.348 g (6 mM, 1 eq) of1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (M_(w)=391.36, Aldrich15033) was added and after 4 hrs DCM was evaporated.

C18:1-norArg-C18:1 (M_(w)=674.1) To the oily residue from the previousreaction 100 ml of 95% TFA/DCM 2.5% TIS was added and after 3 hrssolvent was evaporated. Crude product was purified on TELEDYNE IscoCombiFlash R_(f) instrument using 48 g normal phase silica gel column,100% DCM for 3 CV(column volume) and 0-20% MeOH for 10 CV, detection 214nm, flow 45 ml/min. DCM/MeOH was evaporated and residue was precipitatedby 0.1M HCl. Yield: 3.2 g.

Example 21 Preparation of C10-[4-Pal(N—CH₃)]—C104-(3-(decylamino)-3-oxo-2-decanamidopropyl)-1-methylpyridinium chloride

Fmoc-4-Pal-resin. To 3 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 30ml reaction vessel for solid phase synthesis, 2.27 g (5.85 mmol, 1.5 eq)of Fmoc-4-Pal-OH (Fmoc-4-Pyridinylalanine, M_(w)=388.42, AdvancedChemTech, FX4140) and 2.03 ml (11.7 mmol, 3.0 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added. 4-Pal-resin. After 2 hrs the resin waswashed 3× with DCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmocgroup was deprotected with 30 ml of 20% piperidine/DMF for 30 min.

C10-4-Pal-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.48 g (11.7 mmol)of decanoic acid (Sigma, M_(w)=127.27), 5.54 g (11.7 mmol) of HCTU(M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2, d=0.74) in30 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C10-4-Pal-OH (M_(w)=320.46) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C10-4-Pal-C10 (M_(w)=459.76). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.32 ml (11.7mmol) of C10-amine (Sigma, M_(w)=157.3, d=0.792), 5.54 g (11.7 mmol) ofHCTU (M_(w)=473.7) and 2.23 ml (12.87 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF were added. After 1 hr 100 ml of AcOEt was addedand organic layer was washed in separatory funnel with 3×0.5 M HCl,3×10% NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated. Residue was dissolved in AcCN/0.5 M HCl 1:1 and purified byRP_Akta Explorer on C18 Phenomenex column (Phenomenex RP, 250×21.2 mm,Serial No.: 234236-1, Column volume 83 ml) and eluted with 30-100%acetonitrile gradient using water as mobile phase within 2 CV; λ=215 nm.Acetonitrile was evaporated and the product was lyophilized.

C10-4-Pal(Me)-C10 (M_(w)=474.76). Methylation was carried out insolution. To 0.4 g (0.87 mM) of C10-4-Pal-C10 (M_(w)=459.76) dissolvedin 20 ml of THF, 83 μl (0.87 mmol) of dimethyl sulfate (Sigma,M_(w)=126.13, d=1.325) was added. After overnight reaction the sameamount of dimethyl sulfate was added and after 1 hr the product wasprecipitated with 0.5 M HCl/AcCN mixture.

Example 22 Preparation of C12-[4-Pal(N—CH₃)]—C124-(3-(dodecylamino)-3-oxo-2-dodecanamidopropyl)-1-methylpyridiniumchloride

Fmoc-4-Pal-resin. To 6.5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 70 ml of dry DCM in 200ml reaction vessel for solid phase synthesis, 3.5 g (9.01 mmol, 1.5 eq)of Fmoc-4-Pal-OH (Fmoc-4-Pyridinylalanine, M_(w)=388.42, AdvancedChemTech, FX4140) and 3.132 ml (18 mmol, 2.2 eq) of DIPEA (Aldrich,M_(w)=129.2, d=0.74) were added.

4-Pal-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected 2* with 70ml of 20% piperidine/DMF for 15 min.

C12-4-Pal-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 2.549 g (12.675mmol, 1.5 eq) of lauric acid (Sigma, M_(w)=200.32), 5.3 g (12.675 mmol)of HCTU (M_(w)=417.7) and 2.2 ml (12.675 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 70 ml of DMF were added.

After 1 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C12-4-Pal-OH (M_(w)=346.7) was cleaved from the resin by 1% TFA/DCM(5×30 ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH)and solvent was evaporated.

C12-4-Pal-C12 (M_(w)=515.7). Second coupling was carried out insolution. To the oily residue from the previous reaction 2.3 g (12 mmol)of EDC*HCl (M_(w)=191.7), 1.86 g (12 mmol) of HOBt*H₂O (M_(w)=153.1) and10.44 ml (60 mmol, 5 eq) of DIPEA (M_(w)=129.2, d=0.74) in 70 ml of DCMwere added. Preactivation step was carried out for 20 min and then 2.22g (12 mmol) of C12-amine (Sigma, M_(w)=185.36) was added. Afterovernight reaction the organic layer was washed in separatory funnelwith 3×0.5 M HCl, 3×10% NaCO₃ and 3×NaCl. DCM was dried with anhydrousMgSO₄ and evaporated. The product was purified on TELEDYNE IscoCombiFlash R_(f) instrument, 40 g normal phase silica gel column, 100%DCM for 5 CV(column volume) and 0-5% MeOH for 10 CV, detection 254 nm,flow 40 ml/min.

C12-4-Pal(Me)-C12 (M_(w)=530.8). Methylation was carried out insolution. To 4 g (7.75 mM) of C12-4-Pal-C12 (M_(w)=415.7) dissolved in50 ml of THF, 1.1 ml (11.625 mmol) of dimethyl sulfate (Sigma,M_(w)=126.13, d=1.325) was added. After overnight reaction 0.5 ml ofdimethyl sulfate was added and after 1 hr the solvent was evaporated andthe product was precipitated with 0.5 M HCl.

Example 23 Preparation of C18oleic-[4-Pal]-C16N-(1-oxo-3-(pyridin-4-yl)-1-(hexadecylamino)propan-2-yl)octadec-9-enamide

Fmoc-4-Pal-resin. To 5 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in 50ml reaction vessel for solid phase synthesis 3.787 g (9.75 mmol, 1.5 eq)of Fmoc-4-Pal-OH (M_(w)=388.42, Advanced ChemTech) and 1.7 ml (9.75mmol, 1.5 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.4-Pal-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected with 40 mlof 20% piperidine/DMF 2 times for 15 min.

C18oleic-4-Pal-resin. After Fmoc deprotection the resin was washed with3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g (13 mmol) ofoleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol) of HCTU(M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2, d=0.74) in 50ml of DMF were added.

After 2 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18oleic-4-Pal-OH (M_(w)=430.62) was cleaved from the resin by 1%TFA/DCM (5×50 ml for 2 min was filtered to flask with 2 ml 10%pyridine/MeOH) and solvent was evaporated.

C18oleic-4-Pal-C16 (M_(w)=654.06). Second coupling was carried out insolution. To the oily residue from the previous reaction, 2.354 g (9.75mmol) of C16-amine (Sigma, M_(w)=241.46), 4.033 g (9.75 mmol) of HCTU(M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA (M_(w)=129.2, d=0.74) in50 ml of DMF were added. After 4 hr 100 ml of AcOEt was added andorganic layer was washed in separatory funnel with 3×0.5 M HCl, 3×10%NaCO₃ and 3×NaCl. AcOEt layer was dried with anhydrous MgSO₄ andevaporated. Crude product was purified on TELEDYNE Isco CombiFlash R_(f)instrument using 48 g normal phase silica gel column, 100% DCM for 3CV(column volume) and 0-20% MeOH for 10 CV, detection 214 nm, flow 45ml/min. DCM/MeOH was evaporated and residue was precipitated by 0.1MHCl. Yield: 0.75 g.

Example 24 Preparation of (C18oleic)-(1-CH₃—His)-NH—(C16alkyl)N-(3-(1-methyl-1H-imidazol-4-yl)-1-oxo-1-(hexadecylamino)propan-2-yl)octadec-9-enamide

Fmoc-His(1-Me)-resin. To 1.7 g of 2-chlorotrityl chloride resin with 1.3mmol/g substitution (Novabiochem, 01-64-0114) in 30 ml of dry DCM in 60ml reaction vessel for solid phase synthesis, 1 g (2.5 mmol, 1.2 eq) ofFmoc-His(1-Me)-OH (M_(w)=391.43, ChemImpex) and 0.435 ml (2.5 mmol, 1.2eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) were added.

His(1-Me)-resin. After 2 hrs the resin was washed 3× with DCM/MeOH/DIPEA(17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group was deprotected 2 timeswith 50 ml of 20% piperidine/DMF for 15 min.

C18_(oleic)-His(1-Me)-resin. After Fmoc deprotection the resin waswashed with 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 1.247g (4.4 mmol) of oleic acid (Sigma, M_(w)=282.47; d=0.891), 1.82 g (4.4mmol) of HCTU (M_(w)=413.7) and 0.765 ml (9.1 mmol) of DIPEA(M_(w)=129.2, d=0.74) in 50 ml of DMF were added.

After 2 hr of reaction the resin was washed with 3×DCM, 2×MeOH and 3×DCMand progress of reaction was checked by Kaiser test which was negative(no free amine groups present).

C18_(oleic)-His(1-Me)-OH was cleaved from the resin by 1% TFA/DCM (5×50ml for 2 min was filtered to flask with 2 ml 10% pyridine/MeOH) andsolvent was evaporated.

C18_(oleic)-His(1-Me)-C16 (M_(w)=657.07) Second coupling was carried outin solution. To the oily residue from the previous step 0.604 g (2.5mmol) of C16 amine (Sigma), 1.034 g (2.5 mmol) of HCTU (M_(w)=413.7) and0.435 ml (2.5 mmol) of DIPEA (M_(w)=129.2, d=0.74) in 50 ml of DMF wereadded. After overnight reaction organic layer was diluted with aceticacetate and washed in separatory funnel with 3×0.5 M HCl, 3×10% NaCO₃and 3×NaCl.

AcOEt layer was dried with anhydrous MgSO₄ and evaporated. The crudeproduct was purified on TELEDYNE Isco CombiFlash R_(f) instrument, 40 gnormal phase silica gel column, 100% DCM for 5 CV(column volume) and0-20% MeOH for 10 CV, detection 214 nm, flow 40 ml/min. Purified productwas precipitated with 0.1 M HCl. Yield: 1 g.

Example 25 Preparation of (C18oleic)-(3′,5′-diiodo-Tyr)-NH—(C16alkyl)(E)-N-(1-(hexadecylamino)-3-(4-hydroxy-3,5-diiodophenyl)-1-oxopropan-2-yl)octadec-9-enamide

Proposed Preparation.

Fmoc-Tyr(3′,5′-diI)-resin. To 5 g of 2-chlorotrityl chloride resin with1.3 mmol/g substitution (Novabiochem, 01-64-0114) in 50 ml of dry DCM in50 ml reaction vessel for solid phase synthesis, 6.388 g (9.75 mmol, 1.5eq) of Fmoc-Tyr(3′,5′-diI)—OH (M_(w)=655.2, AnaSpec) and 1.69 ml (9.75mmol, 1.5 eq) of DIPEA (Aldrich, M_(w)=129.2, d=0.74) to be added.

Tyr(3′,5′-diI)-resin. After 3 hrs the resin to be washed 3× withDCM/MeOH/DIPEA (17:2:1), 3×DCM, 2×DMF, 3×DCM and Fmoc group to bedeprotected with 40 ml of 20% piperidine/DMF 2 times for 30 min.

C18oleic-Tyr(3′,5′-diI)-resin. After Fmoc deprotection the resin to bewashed with 3×DCM, 2×MeOH and 3×DCM and for the coupling reaction 3.7 g(13 mmol) of oleic acid (Sigma, M_(w)=282.47, d=0.891), 5.37 g (13 mmol)of HCTU (M_(w)=413.7) and 2.62 ml (13 mmol) of DIPEA (M_(w)=129.2,d=0.74) in 50 ml of DMF to be added.

After 3 hr of reaction the resin to be washed with 3×DCM, 2×MeOH and3×DCM and progress of reaction to be checked by Kaiser test.

C18oleic-Tyr(3′,5′-diI)—OH (M_(w)=697.43) to be cleaved from the resinby 1% TFA/DCM (5×50 ml for 2 min to be filtered to flask with 2 ml 10%pyridine/MeOH) and solvent to be evaporated.

C18oleic-Tyr(3′,5′-diI)—C16 (M_(w)=920.38). Second coupling to becarried out in solution. To the oily residue from the previous reaction,2.354 g (9.75 mmol) of C16-amine (Sigma, M_(w)=241.46), 4.033 g (9.75mmol) of HCTU (M_(w)=413.7) and 1.69 ml (9.75 mmol) of DIPEA(M_(w)=129.2, d=0.74) in 50 ml of DMF to be added. After 4 hr 100 ml ofAcOEt to be added and organic layer to be washed in separatory funnelwith 3×0.5 M HCl, 3×10% NaCO₃ and 3×NaCl. AcOEt layer to be dried withanhydrous MgSO₄ and evaporated. Crude product to be purified on TELEDYNEIsco CombiFlash R_(f) instrument using 48 g normal phase silica gelcolumn, 100% DCM for 3 CV(column volume) and 0-20% MeOH for 10 CV,detection 214 nm, flow 45 ml/min. DCM/MeOH to be evaporated and residueto be precipitated by mixture of AcCN/H₂O.

Example 26 Example In Vitro Assay for LacZ Gene Expression Knockdown in9 L Cells

9 L/LacZ is a rat gliosarcoma cell line stably expressing the LacZ genethat encodes bacterial galactosidase. LacZ gene knockdown measurementscan be used as a primary activity-based in vitro assay for interferingRNA delivery formulations.

For LacZ gene knockdown measurements, 9 L/LacZ cells were transfectedwith an RNAi formulation, and a β-galactosidase assay was performed oncells harvested at day 3 post transfection. An additional assay wasperformed to quantify protein concentration.

9 L/LacZ cells were plated at 8000 cells/well (96-well) and incubatedovernight in medium. Confluency was about 15-20% at the time oftransfection. Transfection complex was prepared by adding an interferingRNA to OptiMEM™ medium and vortexing, separately adding a deliveryformulation to OptiMEM™ medium and vortexing, and finally mixing theinterfering RNA in medium with the delivery formulation in medium tomake the transfection complex. The medium for incubated cells wasreplaced with fresh no-serum media (OptiMEM™ without serum) andtransfection complex was added to each well. Cells were incubated 5 hrs,then after the addition of 100 microliters complete medium (DMEM plus10% fetal bovine serum) were incubated overnight at 37° C. and 5% CO₂.The next day, 24 hours after transfection, the medium was changed tofresh complete medium and the cells were incubated another 48 hrs at 37°C. and 5% CO₂.

For LacZ gene knockdown, the harvested 9 L/LacZ cells were washed inPBS, lysed in M-PER™ Reagent (Pierce), and incubated at room temperaturefor 15 minutes. Lysate was taken from each well for protein assay with aMicro BCA kit (Pierce, ThermoFisher Scientific) and β-gal assay withAll-in-One™ β-Galactosidase Assay Reagent (Pierce).

Example In Vitro Assay for PPIB Gene Expression Knockdown in A549 Cells

Cyclophilin B (PPIB) gene knockdown measurements can be used as aprimary activity-based in vitro assay for interfering RNA deliveryformulations. Cyclophilin B (PPIB) gene expression knockdown wasmeasured in A549 human alveolar basal epithelial cells. For PPIB geneknockdown measurements, A549 cells were transfected with an interferingRNA formulation, total RNA prepared 24 hours after transfection, andPPIB mRNA assayed by RT-PCR. QRT-PCR of 36B4 (acidic ribosomalphosphoprotein PO) mRNA expression was performed for normalization.

A549 cells were seeded at 7,500 cells/well (96-well) and incubatedovernight in medium. Confluency was about 50% at the time oftransfection. Transfection complex was prepared by adding an interferingRNA to medium (OptiMEM™) and vortexing, separately adding a deliveryformulation to medium (OptiMEM™) and vortexing, and finally mixing theinterfering RNA in medium with the delivery formulation in medium andincubating 20 minutes at room temperature to make the transfectioncomplex. The medium for incubated cells was replaced with fresh OptiMEM™and transfection complex was added to each well. Cells were incubatedfor 5 hrs at 37° C. and 5% CO₂, then complete medium was added (to afinal fetal bovine serum concentration 10%) and incubation continueduntil 24 hours post-transfection.

For PPIB gene knockdown cells were lysed and RNA prepared (Invisorb RNACell HTS 96-Kit/C, Invitek, Berlin, or RNeasy 96 Kit, Qiagen).Quantitative RT-PCR was performed using One-Step qRT-PCR kit(Invitrogen) on a DNA Engine Opticon2 thermal cycler (BioRad).

Primers used for PPIB were: (SEQ ID NO: 1) 5′-GGCTCCCAGTTCTTCATCAC-3′(forward) and (SEQ ID NO: 2) 5′-CCTTCCGCACCACCTC-3′ (reverse) with(SEQ ID NO: 3) 5′-FAM-CTAGATGGCAAGCATGTGGTGTTTGG-TAMRA-3′ for the probe.For 36B4, primers were: (SEQ ID NO: 4) 5′-TCTATCATCAACGGGTACAAACGA-3′(forward) and (SEQ ID NO: 5) 5′-CTTTTCAGCAAGTGGGAAGGTG-3′ (reverse) with(SEQ ID NO: 6) 5′-FAM-CCTGGCCTTGTCTGTGGAGACGGATTA-TAMRA-3′for the probe.

Example In Vivo Assay for Influenza Viral Titer Knockdown in Mouse

Influenza viral titer knockdown measurements in mice can be used as anin vivo gauge of efficacy for interfering RNA amino acid lipid deliveryformulations.

In this assay, typically 50 uL of a dsRNA amino acid lipid formulation,or PBS for a control group, was administered intranasally in 7-9 weekold Balb/C mice anesthetized with ketamine/xylazine. Daily dosing wasperformed for 3 consecutive days on days −2, −1, and 0. Infection wasinduced 4 hours after the last dosing.

Influenza infection was induced with Influenza A/Puerto Rico/8/34 (PR8,subtype H1N1). For infection, 50 μl of 20 pfu PR8 diluted in 0.3%BSA/1×PBS/PS was administered intranasally into mice anesthetized withketamine/xylazine. 48 hours after infection, the lungs were harvestedand homogenized in 600 uL 0.3% BSA/1×PBS/PS. The homogenates were frozenand thawed twice to release the virus. A TCID50 assay (Tissue-CultureInfectious Dose 50) was performed to titer virus in lung homogenates.Flat-bottom, 96-well plates were seeded with 2×10⁴ MDCK cells per well,and 24 hours later, the serum-containing medium was removed. 30 uL oflung homogenates, either undiluted or diluted from 10- to 10⁷-fold (in10-fold steps), was added into quadruplicate wells. After incubation for1 hr, 170 μl of infection medium (DMEM/0.3% BSA/10 mM HEPES/PS)containing 4 g/ml trypsin was added to each well. After incubation for48 hours at 37° C., the presence or absence of virus in the culturesupernatants was determined by hemagglutination of chicken red bloodcells. The virus titers were estimated using the Spearman and Karberformula.

Example SYBR™ Gold Assay for siRNA Concentrations

The concentration of dsRNA in a formulation can be determined by SYBR™Gold assay as follows: 10 ul of dsRNA formulation is added to 100 ulMeOH and incubated for 5 minutes at 55° C. 50 ul Heparin (200 mg/ml) isadded, and the solution is incubated for 5 minutes at 55° C. 790 ul PBS(phosphate buffered saline) is added, and the sample is spun down inmicrofuge to pelletize. A 90 ul sample of the pellet is incubated with10 ul SYBR™ Gold reagent (Molecular Probes, Eugene, Oreg.). Fluorescenceis read at Em 535 nm with excitation at 495 nm.

Example RNA Isolation and Quantitative RT-PCR Assay for Knockdown ofApoB Message

For this housekeeping gene protocol, pulmonary or systemic, on Day 1C57/B6 or Balb/C female mice age 8-10 weeks (5 mice per group) weredosed with 50 ul or 200 uL of dsRNA formulation by intranasal orintravenous route of administration, respectively. Anesthetic wasketamine/xylazine (IP—0.2 ml dose/mouse). On Day 2 whole lung wasisolated from mice either 1 day post dosing or next day following 3consecutive days of dosing for pulmonary model or lung, liver, kidney,spleen, heart and/or whole blood for systemic model. Tissues were placedin a 24 well plate containing 2 ml RNALATER RNA Stabilization Reagent(Qiagen 76106). Plates were placed on dry ice to freeze immediately, andstored at −80° C. until homogenate was prepared.

Total RNA was extracted from tissue samples stored in RNALATER solutionat the time of necropsy. The isolation of total RNA was done using theInvitrogen PURELINK 96 RNA Isolation Kit according to the manufacturer'sprotocol for the isolation of mammalian tissue. These total RNA isolateswere then quantified using the NanoDrop spectrophotometer, and thenquality was assured using the Agilent Bioanalyzer 2100 system todetermine RNA integrity. After the quality and quantity of the total RNAwas determined, the samples were normalized to equal concentrations and50-100 ng of total RNA was synthesized into cDNA using the AppliedBiosystems High Capacity cDNA Archive Kit according to themanufacturer's protocol. The cDNA was then rechecked for concentrationusing the Nanodrop and then diluted 1:10 for qRT-PCR analysis.

The gene expression analysis using qRT-PCR was done using the cDNAsamples and SYBR green technology with final primer concentrations of200 nM. The samples were run using the Quanta PerfectCta SYBR GreenFastMix, ROX using a 10 μL reaction volume. The samples were run using a384-well plate format on the Applied Biosystems 7900HT platform usingfast cycling conditions. Data was then exported from the SDS 2.3software using a threshold value of 0.2. These were formatted foranalysis using QBASE software of algorithms entered by the user intoExcel. Genes chosen for endogenous controls were based on geNormanalysis. For mouse liver samples GAPDH and PPIA and TBP have been shownto be very good stable normalizers for gene expression analysis.

Serum Cholesterol Assay

The purpose of this assay was to ascertain the blood serum cholesterollevels in mouse blood 48 hours after dosing with a dsRNA. InvitrogenAmplex Red Cholesterol Assay Kit (cat#A12216) was used. Mouse wholeblood was drawn and placed in Serum Separator Tubes (SST-BD cat#365956), then centrifuged to separate the serum from the rest of theblood. The serum was diluted in DI-H₂0 1:40, then diluted a further 1:5(total of 1:200) in the 1× Reaction buffer from the Amplex assay kit.Standards were made from the cholesterol reference standard, suppliedwith the kit, at concentrations of 20 ug/ml, 10 ug/ml, 8 ug/ml, 6 ug/ml,4 ug/ml, 2 ug/ml and 1 ug/ml. Samples, standards and blanks were addedto a flat bottomed black 96-well plate (Costar Catalogue #3916). TheAmplex assay mixture was made and added to the assay wells. After atleast 30 minutes of incubation at 37° C., the plate was read using theMolecular Devices SpectraMax M5. The “End point” protocol was selected,and the excitation range was set for 530-560 (preferably 544 nM), andthe emission detection at ˜590. Once the plate was read the data wasexported to excel, where the Percent control, percent reduction andtotal blood serum cholesterol in ug/ml was calculated.

Example 27

The structures of some double-stranded RNAs (dsRNA) of this disclosureare shown in Table 1.

TABLE 1 Double-stranded RNAs RNA SEQUENCES DX4227 (SEQ ID NO: 7) ApoBSense 5′-GGAAUC_(m)U_(m)UA_(m)UA_(m)U_(m)U_(m)UGAUC_(m)CAsA-3′(SEQ ID NO: 8)Antisense 5′-_(m)U_(m)UGGAU_(m)CAAA_(m)UA_(m)UAAGA_(m)UUC_(m)Cs_(m)CsU-3′DX3030 (SEQ ID NO: 9) Influenza Sense 5′-GGAUCUUAUUUCUUCGGAGACAAdTdG-3′(SEQ ID NO: 10) Antisense 5′-CAUUGUCUCCGAAGAAAUAAGAUCCUU-3′ DX2816(SEQ ID NO: 11) Non-target Sense 5′-UUCUCCGAACGUGUCACGUdTdT-3′ Qneg(SEQ ID NO: 12) Antisense 5′-ACGUGACACGUUCGGAGAAdTdT-3′ DX2940(SEQ ID NO: 13) LacZ Sense 5′-CUACACAAAUCAGCGAUUUdTdT-3′ (SEQ ID NO: 14)Antisense 5′-AAAUCGCUGAUUUGUGUAGdTdC-3′ DX2742 (SEQ ID NO: 15) PPIBSense 5′-GGAAAGACUGUUCCAAAAAUU-3′ MoCypB (SEQ ID NO: 16)Antisense 5′-UUUUUGGAACAGUCUUUCCUU-3′ DX 2744 (SEQ ID NO: 17) G1498Sense 5′-GGAUCUUAUUUCUUCGGAGdTdT-3′ influenza (SEQ ID NO: 18)Antisense 5′-CUCCGAAGAAAUAAGAUCCdTdT-3′ DX 2918 (SEQ ID NO: 19) Inm4Sense 5′-CCGTCAGCCGATTTGCTATTT-3′ TNFa (SEQ ID NO: 20) modifiedAntisense 5′-p-AUAGCAAATCGGCTGACGGTT-3′

In Table 1, “mU” represents 2′-O-methyl uridine, “mC” represents2′-O-methyl cytidine, and “s” represents a phosphorothioate linkage.

Example 28

Active RNA formulations of this disclosure can be prepared by dissolvingan interfering RNA in buffer or cell culture medium and vortexing,separately admixing a delivery formulation with buffer or cell culturemedium and vortexing, and finally admixing the interfering RNA mixturewith the delivery formulation mixture to make an active RNAitransfection formulation.

To prepare a delivery formulation, amino acid lipids along with otherlipids and/or excipients can be solubilized in CHCl₃/MeOH, dried downunder N₂, and hydrated in 10 mM HEPES with 5% dextrose at pH 7.4. Themixture can be sonicated, or extruded, dialyzed, and/or tangential flowfiltered.

Various dilution methods known in the art can also be used to prepareactive RNA formulations of this disclosure.

An exemplary interfering RNA formulation of this disclosure is shown inTable 2. In this example, the amino acid lipid designated C8-Arg-C8provides its own formulation for intracellular delivery of aninterfering siRNA therapeutic. The amount of amino acid lipid is givenas the mole percentage of delivery components, not including the activeRNA agent. The designation “C16-Arg-C14” refers to(C15alkyl)-(C═O)-Arg-NH—(C14alkyl) which is the same as(C16acyl)-Arg-NH—(C14alkyl).

TABLE 2 dsRNA Formulation Component Amount dsRNA 50 nM C16-Arg-C14 aminoacid lipid 100 mole %

An exemplary interfering RNA formulation of this disclosure is shown inTable 3. In this example, the amino acid lipid designated C14-norArg-C14provides its own formulation for intracellular delivery of aninterfering siRNA therapeutic. The amount of amino acid lipid is givenas the mole percentage of delivery components, not including the activeRNA agent. The designation “C14-norArg-C14” refers to(C13alkyl)-(C═O)-norArg-NH—(C14alkyl) which is the same as(C14acyl)-norArg-NH—(C14alkyl).

TABLE 3 dsRNA Formulation Component Amount dsRNA 50 nM C14-norArg-C14amino acid lipid 100 mole %

An exemplary RNAi formulation of this disclosure is shown in Table 4. Inthis example, the amino acid lipid designated C10-norArg-C10 is combinedwith a non-cationic lipid in a co-delivery formulation. The designation“C10-norArg-C10” refers to (C9alkyl)-(C═O)-norArg-NH—(C10alkyl) which isthe same as (C10acyl)-norArg-NH—(C12alkyl).

TABLE 4 dsRNA Formulation Component Amount dsRNA DX3030 50 nMC10-norArg-C10 amino acid lipid 50 mole % DOPE 50 mole %

An exemplary RNAi formulation of this disclosure is shown in Table 5. Inthis example, the amino acid lipid designated C12-homoArg-C12 iscombined with a cationic lipid, a non-cationic lipid, and a pegylatedlipid in a multicomponent delivery formulation. The designation“C12-homoArg-C12” refers to (C11 alkyl)-(C═O)-homoArg-NH—(C12alkyl)which is the same as (C12acyl)-homoArg-NH—(C12alkyl).

TABLE 5 dsRNA Formulation Component Amount dsRNA 25 nM C12-norArg-C12 30mole % DSPC 20 mole % Cholesterol 49 mole % DSPE-PEG2000 1 mole %

An exemplary interfering-RNA emulsion composition of this disclosure isshown in Table 6. In this example, the cationic amino acid lipid isdesignated C14-norArg-C14.

TABLE 6 dsRNA Emulsion Component Amount dsRNA 25 nM, N/P 4C14-norArg-C14 11% (w/w of oil phase); dioleoylphosphatidylethanolamine1:1 molar ratio of cationic lipid to emulsifier LABRAFAC PG 89% (w/w ofoil phase) water 80%

An exemplary interfering-RNA dispersion composition of this disclosureis shown in Table 7. In this example, the cationic amino acid lipid isdesignated C14-norArg-C14.

TABLE 7 dsRNA Dispersion Composition Component Amount dsRNA 25 nM, N/P 4C14-norArg-C14 12% (w/w of lipid/dispersant phase) LABRASOL 88% (w/w oflipid/dispersant phase) water 80%

Example 29

Example liposomal formulations of interfering RNA compositions of thisdisclosure are shown in Table 8.

TABLE 8 Example Formulations Composition (mol %)C18-norArg(NH₃Cl)—C18/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C16-norArg(NH₃Cl)—C16/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C14-norArg(NH₃Cl)—C14/DMPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C12-Arg(NH₃Cl)—C12/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C10-Arg(NH₃Cl)—C10/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C8-Arg(NH₃Cl)—C8/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C10-D-Arg(NH₃Cl)—C18/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C12-D-Arg(NH₃Cl)—C16/DPPE-PEG5k/DSPC/chol. (30; 1; 20; 49)C14-D-Arg(NH₃Cl)—C14/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C16-homoArg(NH₃Cl)—C12/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C18-homoArg(NH₃Cl)—C10/DMPE-PEG5k/DSPC/chol. (30; 1; 20; 49)C12-homoArg(NH₃Cl)—C12/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C14-nornorArg(NH₃Cl)—C14/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)C16-nornorArg(NH₃Cl)—C16/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)

Example 30 Particle Size of RNA Delivery Compositions

Examples of liposomal amino acid lipid interfering RNA formulations areshown in Table 9. The formulations of Table 9 each had an N/P of 1.8 andexhibited particle sizes from 106-139 nm with dispersity values of about0.08 to 0.19.

TABLE 9 Particle Size of RNA Delivery Compositions Particle Composition(mol %) N/P Size (nm) PDI C12-norArg(NH₃Cl)—C12/ 1.8 139 0.19DSPE-PEG2k/DSPC/CHOL (30; 1; 20; 49) C12-Arg(NH₃Cl)—C12/ 1.8 106 0.08DSPE-PEG2k/DSPC/CHOL (30; 1; 20; 49) C12-D-Arg(NH₃Cl)—C12/ 1.8 112 0.19DSPE-PEG2k/DSPC/CHOL (30; 1; 20; 49) C12-homoArg(NH₃Cl)—C12/ 1.8 1190.15 DSPE-PEG2k/DSPC/CHOL (30/1/20/49)

Examples of amino acid lipid interfering RNA formulations are shown inTable 10. The formulations of Table 10 had N/P ratios of from 1.5 to 1.9and exhibited particle sizes from 140-148 nm with dispersity values ofabout 0.18 to 0.30.

TABLE 10 Particle Size of RNA Delivery Compositions Particle CompositionN/P Size (nm) PDI C12-nor-Arg(NH₃Cl)—C12/ 1.51 148.3 0.18DSPE-PEG2k/DSPC/CHOL (30/1/20/49) C12-Arg(NH₃Cl)—C12/ 1.51 148.3 0.18DSPE-PEG2k/DSPC/CHOL (30/1/20/49) C12-D-Arg(NH₃Cl)—C12/ 1.51 148.3 0.18DSPE-PEG2k/DSPC/CHOL (30/1/20/49) C12-homoArg(NH₃Cl)—C12/ 1.89 140.30.30 DSPE-PEG2k/DSPC/CHOL (30/1/20/49)

A transmission electron micrograph obtained on a JEOL 1230 TEM of aliposomal embodiment of this invention is shown in FIG. 2. FIG. 2 showsspherical lipid bilayer vesicle particles formed with the amino acidlipid C12-norArg-C12. The length marker of the micrograph is 0.5micrometer, and the sample was stained with 3% uranyl acetate. The lipidportion of the liposomal formulation was [C12-norArg(NH₃⁺Cl⁻)—C12/DSPC/cholesterol/DSPE-PEG-2000] in the amounts of[30%/20%/49%/1%] respectively, as a percent weight/weight of totallipid. The liposomes contained the antiinfluenza-active dicer substratedsRNA DX3030.

Example 31 LacZ Gene Knockdown Activity In Vitro for Amino Acid LipidRNAi Binary Compositions

Amino acid lipids provided effective interfering-RNA deliverycomposition as shown in Table 11 by a LacZ activity-based in vitroassay. The results in Table 11 showed that gene knockdown by aninterfering-RNA in an amino acid lipid composition can exceed thatobtained with RNAIMAX (Invitrogen).

TABLE 11 LacZ Knockdown for Amino Acid Lipid Interfering RNAFormulations Composition % Knockdown vs. Qneg C8-norArg-C8:DOPE (1:1) 88C10-norArg-C10:DOPE (1:1) 89 C12-norArg-C12:DOPE (1:1) 88C8-L-Arg-C8:DOPE (1:1) 16 C10-L-Arg-C10:DOPE (1:1) 82 C12-L-Arg-C12:DOPE(1:1) 100 C8-D-Arg-C8:DOPE (1:1) 40 C10-D-Arg-C10:DOPE (1:1) 82C12-D-Arg-C12:DOPE (1:1) 80 C8-homoArg-C8:DOPE (1:1) 52C10-homoArg-C10:DOPE (1:1) 100 C12-homoArg-C12:DOPE (1:1) 51C8-nornorArg-C8:DOPE (1:1) 78 C10-nornorArg-C10:DOPE (1:1) 78C8-norArg-C8:DPhPE (1:1) 43 C10-norArg-C10:DPhPE (1:1) 73 RNAI-MAX 92

In Table 11, the final concentration of dsRNA was 100 nM and the N/Pratio was 1.8 for each composition.

Example 32

LacZ Gene Knockdown In Vitro for Amino Acid Lipid RNAi LiposomalCompositions Amino acid lipids provided effective interfering-RNAdelivery compositions as shown in Table 12 by a LacZ activity-based invitro assay.

TABLE 12 Normalized LacZ Gene Knockdown for Amino Acid Lipid RNAiCompositions % Knock- Composition downC12-norArg(NH₃Cl)—C12/DPhPE/DSPE-PEG (50/49/1) 60C12-Arg(NH₃Cl)—C12/DPhPE/DSPE-PEG (50/49/1) 94C12-D-Arg(NH₃Cl)—C12/DPhPE/DSPE-PEG (50/49/1) 86C12-homoArg(NH₃Cl)—C12/DPhPE/DSPE-PEG (50/49/1) 81 RNAIMAX 82

The normalized LacZ knockdown results in Table 12 showed that at least60% knockdown was observed for all amino acid lipid compositions. Theresults in Table 12 showed that gene knockdown by an interfering-RNA inan amino acid lipid composition can exceed that obtained with RNAIMAX.

Example 33 Gene Knockdown Concentration Response In Vitro for Amino AcidLipid RNAi Compositions

Gene knockdown activity for several ternary amino acid lipid RNAicompositions was determined in vitro.

Amino acid lipids provided efficacious interfering-RNA deliverycomposition as shown in FIG. 3 by a PPIB knockdown activity-based invitro assay in A549 cells. The results in FIG. 3 showed that geneknockdown by an interfering-RNA in an amino acid lipid composition, asrepresented by normalized PPIB values, can exceed that obtained withRNAIMAX.

In FIG. 3 is shown the concentration response of the normalized PPIBvalues for two amino acid lipid formulations compared to results forRNAIMAX. Formulation 1 in FIG. 3 was [C12-norArg(NH₃Cl)—C12/DOPE/CHOL(50/32/18)] and Formulation 2 was [C12-norArg(NH₃Cl)—C12/CHEMS/DLPE(50/32/18)].

Amino acid lipids provided efficacious interfering-RNA deliverycomposition as shown in FIG. 4 by a LacZ knockdown activity-based invitro assay in 9 L cells. The results in FIG. 4 showed that geneknockdown by an interfering-RNA in an amino acid lipid composition, asrepresented by normalized beta-galactosidase values, can exceed thatobtained with RNAIMAX.

In FIG. 4 is shown the concentration response of the normalizedbeta-galactosidase values for two amino acid lipid formulations comparedto results for RNAIMAX. Formulation 1 in FIG. 4 was[C12-norArg(NH₃Cl)—C12/DOPE/CHOL (50/32/18)] and Formulation 2 was[C12-norArg(NH₃Cl)—C12/CHEMS/DLPE (50/32/18)].

The gene knockdown activity data in vitro for several amino acid lipidformulations containing three lipid components are summarized in Table13.

TABLE 13 Gene Knockdown for Amino Acid Lipid RNAi Compositions In vitro% Knockdown (vs QNeg) dsRNA A549 9L Formulation (nM) PPIB LacZC12norArgC12/CHEMS/chol. (50/32/18) 25 85 95 (N/P = 1.8) 10 74 74 1 9 49C12norArgC12/DOPE/chol. (50/32/18) 25 92 97 (N/P = 5.0) 10 93 91 1 34 30C12norArgC12/CHEMS/DLPE (50/32/18) 25 98 97 (N/P = 1.8) 10 97 94 1 23 39C12norArg/chol./DLPE (50/32/18) 25 78 83 (N/P = 5.0) 10 43 57 1 2 24RNAIMAX 25 87 89 10 42 67 1 7 16

As shown in Table 13, the results obtained at a concentration of 10 nMdsRNA showed that these exemplary amino acid lipid formulationscontaining C12-norArg-C12 provided gene knockdown activity in vitro thatexceeded results obtained with RNAIMAX.

The gene knockdown activity data in vitro for some amino acid lipidformulations containing three lipid components obtained with and withoutserum present (fetal bovine serum) are summarized in Table 14.

TABLE 14 PPIB gene knockdown for amino acid lipid RNAi compositions invitro with and without serum % Knockdown (vs Qneg) dsRNA w/ w/oFormulation N:P (nM) Serum Serum C12-norArg-C12/CHEMS/chol. 4 25 94 91 42.5 69 83 4 0.25 42 23 C12-norArg-C12/CHEMS/chol. 4 25 96 95 4 2.5 95 964 0.25 65 47 RNAIMAX — 25 98 97 (fixed amount per preparation) — 2.5 9794 — 0.25 56 48

As shown in Table 14, exemplary amino acid lipid formulations containingC12-norArg-C12 provided high PPIB gene knockdown activity in vitro withand without serum present. These amino acid lipid formulations were madeby various rehydration and dilution methods.

The gene knockdown activity data in vitro for some amino acid lipidformulations containing three and four lipid components are summarizedin Table 15.

TABLE 15 LacZ Gene Knockdown (KD) for Amino Acid Lipid RNAi Compositionsin vitro % KD %(w/w) of dsRNA vs Qneg Composition lipids (nM) 9L/LacZC18:1-His(1-Me)—C16/C18:1-  8/42/50 25 90 norArg-C16/chol. 5 83 16/32/5025 91 5 94 C18:1-His(1-Me)—C16/C18:1-  8/42/50 25 94 norArg-C16/chol. 590 16/32/50 25 91 5 91 C18:1-norArg-C16/CHEMS/chol. 50/32/18 25 43 5 5650/32/18 25 72 5 73 C18:1-norArg-C12/CHEMS/chol. 50/32/18 25 90 5 92C18:1-norArg-C8/CHEMS/chol. 50/32/18 25 92 5 95C18:2-norArg-C16/CHEMS/chol. 50/32/18 25 90 5 33 50/20/30 25 92 50/24/2625 93 50/28/22 25 92 C18:1-norArg-C16/CHEMS/ 40/16/34/10 25 79chol./DSPC 40/19/31/10 25 82 40/22/28/10 25 82 40/26/24/10 25 88C18:1-Pal-C16/C18:1-norArg-  4/46/50 25 72 C16/chol.  8/42/50 25 7716/32/50 25 88 32/16/50 25 91

As shown in Table 15, some amino acid lipid formulations containingthree and four components provided high 9 L/LacZ gene knockdown activityin vitro. High 9 L/LacZ knockdown activity was observed for formulationsusing a mixture of amino acid lipids, and for formulations having CHEMSor cholesterol as an additional lipid.

Example 34 ApoB Gene Knockdown Concentration Response In Vitro for AminoAcid Lipid RNAi Compositions

ApoB gene knockdown activity for several four-component amino acid lipidRNAi compositions was determined in vitro.

In FIG. 5 is shown an example of ApoB gene knockdown activity obtainedfrom an in vitro assay in HepG2 cells. The concentration response at 25,2.5, and 0.25 nM RNA of the normalized ApoB mRNA expression values forthree amino acid lipid formulations of an interfering-RNA are shown.Formulation 1 was [non-amino acid cationic lipid/DSPC/chol./DMPE-PEG2k(40/10/48/2)]. Formulation 2 and 3 were both[C18:1-norArg-C16/CHEMS/DLPE/DMPE-PEG2k (50/32/16/2)].

The ApoB dsRNA was DX4227 (ApoB-2 P2098).

A concentration response was obtained for percent ApoB mRNA expressionknockdown in HepG2 cells. For the HepG2 cell screen the protocol was asfollows: On Day 1 the HepG2 cells were refreshed with fresh growthmedium containing 10% FBS, lx non-essential amino acid, and 0.125%sodium bicarbonate in DMEM. On Day 2, 25 microL of the complex was addedto the wells, then 75 microL of HepG2 single cell suspension in OPTIMEMwas added to the wells (15000 cells/well). After 4 hours, 100 ul DMEMwith 20% FBS was added to each well. On Day 3 at 24 hours the cells werelysed, the RNA was prepared, and qRT-PCR was performed for ApoB andrGAPDH mRNA.

The ApoB gene knockdown activity for several four-component amino acidlipid RNAi compositions is summarized in Table 16.

TABLE 16 ApoB Gene Knockdown for Amino Acid Lipid RNAi Compositions invitro N/P pH pH % Knockdown vs UNT Composition 7.4 5.0 100 nM 25 nM 2.5nM 0.25 nM C18:1-norArg-C16/CHEMS/DLPE/DMPE-PEG2k 1.8 4.9 84 93 36 0(50/32/16/2) C18:1-norArg-C16/CHEMS/DMPE/DMPE-PEG2k 0.8 2.1 94 84 0 0(50/32/16/2) C18:1-norArg-C16/CHEMS/DPPE/DMPE-PEG2k 0.8 2.1 96 82 19 0(50/32/16/2) C18:1-norArg-C16/CHEMS/DPLC/DMPE-PEG2k 0.8 2.1 96 89 0 0(50/32/16/2) C18:1-norArg-C16/CHEMS/DSPC/DMPE-PEG2k 0.8 2.1 94 89 37 0(50/32/16/2) C18:1-norArg-C16/CHEMS/DLPE/DMPE-PEG2k 0.8 2.1 95 82 13 13(50/32/16/2) RNAIMAX — — 87 24 24

Example 35 ApoB Gene Knockdown In Vivo for Amino Acid Lipid RNAiCompositions

ApoB gene knockdown activity for some three- and four-component aminoacid lipid RNAi compositions was determined in vivo mouse. The ApoB mRNAreduction activity in vivo is shown in Table 17.

TABLE 17 ApoB Gene Knockdown for Amino Acid Lipid RNAi Compositions invivo % Knockdown Composition In VivoC18:1-norArg-C16/CHEMS/DLPE/DMPE-PEG2k 88 (50/32/16/2)C18:1-norArg-C16/chol./DMPE-PEG2k 45 (50/48/2)C18:1-norArg-C16/CHEMS/chol./DMPE-PEG2k 46 (50/15/33/2)C18:1-norArg-C16/CHEMS/chol./DMPE-PEG2k 88 (50/32/16/2)

The ApoB gene knockdown dose response in vivo mouse was obtained forseveral four-component amino acid lipid RNAi compositions and comparedto mouse serum cholesterol levels. The ApoB mRNA reduction andcorresponding serum cholesterol reduction in vivo is summarized in Table18.

TABLE 18 ApoB Gene Knockdown (KD) Dose Response for Amino Acid LipidRNAi Compositions in vivo ApoB ApoB % Change in mRNA mRNA serum % KD %KD cholesterol Composition (ApoB 9133) (ApoB 12211) (+/−gain/loss)C18:1-norArg-C16/ 72.0 72.6 −7.1 CHEMS/DLPE/DMPE- PEG2k (50/32/16/2)(N/P 1.8, 2 mg/kg) C18:1-norArg-C16/ 35.2 39.6 −4.0 CHEMS/DLPE/DMPE-PEG2k (50/32/16/2) (N/P 1.8, 1 mg/kg) C18:1-norArg-C16/ 17.6 20.6 10.6CHEMS/DLPE/DMPE- PEG2k (50/32/16/2) (N/P 1.8, 0.5 mg/kg)C18:1-norArg-C16/ 69.9 70.6 −38.9 CHEMS/DLPE/DMPE- PEG2k (50/32/16/2)(N/P 0.8, 4 mg/kg) C18:1-norArg-C16/ 46.3 47.8 −14.5 CHEMS/DLPE/DMPE-PEG2k (50/32/16/2) (N/P 0.8, 2 mg/kg) PBS — 1.2 0

Example 36 Antiviral Effects for Amino Acid Lipid RNAi Compositions

Examples of amino acid lipid interfering RNA formulations having fourcomponents are shown in Table 19. The formulations of Table 19 were usedto demonstrate anti-viral activity in vivo in a mouse influenza model.

TABLE 19 Example RNA Delivery and Comparative Formulations dsRNA doseLipid (amt/ dose kg/day) (μmol/ Group Composition dsRNA mg nmol kg/day)1 C12-norArg(NH₃ ⁺Cl⁻)—C12/ DX3030 2 120 36 DSPE-PEG2k/DSPC/chol. (30;1; 20; 49) 2 C12-norArg(NH₃ ⁺Cl⁻)—C12/ Qneg 1.6 120 28.8DSPE-PEG2k/DSPC/chol. DX2816 (30; 1; 20; 49) 3 C14-norArg (NH₃⁺Cl⁻)—C14/ DX3030 2 120 36 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 4C14-norArg (NH₃ ⁺Cl⁻)—C14/ Qneg 1.6 120 28.8 DSPE-PEG2k/DSPC/chol.DX2816 (30; 1; 20; 49) 5 C16- DX3030 2 120 36 norArg (NH₃ ⁺Cl⁻)—C16/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 6 C16-norArg (NH₃ ⁺Cl⁻)—C16/ Qneg1.6 120 28.8 DSPE-PEG2k/DSPC/chol. DX2816 (30; 1; 20; 49) 7 C18-norArg(NH₃ ⁺Cl⁻)—C18/ DX3030 2 120 36 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 8C18-norArg (NH₃ ⁺Cl⁻)—C18/ Qneg 1.6 120 28.8 DSPE-PEG2k/DSPC/chol.DX2816 (30; 1; 20; 49) 9 C18oleic- DX3030 2 120 36 norArg(NH₃ ⁺Cl⁻)—C16/DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 10 C18oleic- Qneg 1.6 120 28.8norArg(NH₃ ⁺Cl⁻)—C16/ DX2816 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 11C12-norArg(NH₃ ⁺Cl⁻)—C12/ NONE — — 36 DSPE-PEG2k/DSPC/chol. (30; 1; 20;49) 12 PBS — — — —

The formulations of Table 19 each had an N/P of 1.8 and exhibitedparticle sizes from 127-183 nm (excepting Groups 7-8) with dispersityvalues of about 0.1 to 0.3, as shown in Table 20.

TABLE 20 Characterization of RNA Delivery and Comparative FormulationsGroup Composition N/P Size (nm) PDI 1 C12-norArg(NH₃ ⁺Cl⁻)—C12/ 1.8151.1 0.315 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 2 C12-norArg(NH₃⁺Cl⁻)—C12/ 1.8 128 0.134 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 3C14-norArg (NH₃ ⁺Cl⁻)—C14/ 1.8 144.5 0.201 DSPE-PEG2k/DSPC/chol. (30; 1;20; 49) 4 C14-norArg (NH₃ ⁺Cl⁻)—C14/ 1.8 126.6 0.114DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 5 C16-norArg (NH₃ ⁺Cl⁻)—C16/ 1.8182.8 0.141 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 6 C16-norArg (NH₃⁺Cl⁻)—C16/ 1.8 174 0.157 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 7C18-norArg (NH₃ ⁺Cl⁻)—C18/ 1.8 977.4 1 DSPE-PEG2k/DSPC/chol. (30; 1; 20;49) 8 C18-norArg (NH₃ ⁺Cl⁻)—C18/ 1.8 625 0.925 DSPE-PEG2k/DSPC/chol.(30; 1; 20; 49) 9 C18oleic-norArg(NH₃ ⁺Cl⁻)—C16/ 1.8 139.3 0.155DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49) 10 C18oleic-norArg(NH₃ ⁺Cl⁻)—C16/1.8 130.2 0.109 DSPE-PEG2k/DSPC/chol. (30; 1; 20; 49)

The degrees of viral titer reduction for the formulations of Table 19are shown in Table 21. Each value for viral titer reduction reflectsdata for eight mice.

TABLE 21 Viral Titer Reduction for RNAi Delivery Formulations FoldReduction Fold Reduction in Viral Titer in Viral Titer Compared toCompared to PBS control PBS control Group dsRNA (averages) (medians) pvalue 1 DX3030 165.0 1698.0 0.0002 2 Qneg 2.1 2.3 0.034 DX2816 3 DX303025.3 100.0 0.002 4 Qneg 3.8 5.6 0.008 DX2816 5 DX3030 13.8 17.8 0.0003 6Qneg 6.3 6.7 0.002 DX2816 7 DX3030 26.7 31.6 0.001 8 Qneg 2.6 5.6 0.037DX2816 9 DX3030 25.7 22.8 0.0002 10 Qneg 20.0 17.8 0.0003 DX2816

In general, the results in Table 21 showed that the interfering-RNAamino acid lipid formulations delivered the interfering-RNA to cells invivo, whereby influenza viral titer was reduced by up to about 1600-foldor greater, relative to PBS as control. Control Groups were #2, 4, 6, 8,and 10, which were dosed with Qneg.

What is claimed is:
 1. A compound comprising the structure shown inFormula I:R³—(C═O)-Xaa-Z—R⁴  Formula I wherein Xaa is any D- or L-amino acidresidue having the formula —NR^(N)—CR¹R²—(C═O)—, or a peptide of n=2-20amino acid residues having the formula —{NR^(N)—CR¹R²—(C═O)}_(n)—,wherein R¹ is independently, for each occurrence, a non-hydrogen,substituted or unsubstituted side chain of an amino acid; R² isindependently, for each occurrence, hydrogen, or an organic groupconsisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, andhaving from 1 to 20 carbon atoms, or C(1-5)alkyl, cycloalkyl,cycloalkylalkyl, C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl, cyano-C(1-5)alkyl,aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl, carboxyl, or hydroxyl, R^(N)is independently, for each occurrence, hydrogen, or an organic groupconsisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, andhaving from 1 to 20 carbon atoms, or C(1-5)alkyl, cycloalkyl,cycloalkylalkyl, C(3-5)alkenyl, C(3-5)alkynyl, C(1-5)alkanoyl,C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl,C(1-5)alkoxy-C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-,C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl, cyano-C(1-5)alkyl,aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl, carboxyl, or hydroxyl, R³ isindependently a lipophilic tail derived from a naturally-occurring orsynthetic lipid, phospholipid, glycolipid, triacylglycerol,glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside,or ganglioside, wherein the tail may contain a steroid; or a substitutedor unsubstituted C(3-22)alkyl, C(6-12)cycloalkyl,C(6-12)cycloalkyl-C(3-22)alkyl, C(3-22)alkenyl, C(3-22)alkynyl,C(3-22)alkoxy, or C(6-12)alkoxy-C(3-22)alkyl; R⁴ is independently alipophilic tail derived from a naturally-occurring or synthetic lipid,phospholipid, glycolipid, triacylglycerol, glycerophospholipid,sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside,wherein the tail may contain a steroid; or a substituted orunsubstituted C(3-22)alkyl, C(6-12)cycloalkyl,C(6-12)cycloalkyl-C(3-22)alkyl, C(3-22)alkenyl, C(3-22)alkynyl,C(3-22)alkoxy, or C(6-12)alkoxy-C(3-22)alkyl; wherein either one of R³and R⁴ is a lipophilic tail as defined above and the other is an aminoacid terminal group, or both R³ and R⁴ are lipophilic tails; the aminoacid terminal group being hydrogen, hydroxyl, amino, or an organicprotective group; Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linkerconsisting of 1-40 atoms selected from hydrogen, carbon, oxygen,nitrogen, and sulfur atoms; and salts thereof.
 2. A compound comprisingthe structure shown in Formula I:R³—(C═O)-Xaa-Z—R⁴  Formula I wherein Xaa is a D- or L-amino acid residuehaving the formula —NR^(N)—CR¹R²—(C═O)—, wherein R¹ is a substituted orunsubstituted basic side chain of an amino acid; R² is hydrogen, orC(1-5)alkyl, R^(N) is hydrogen, or C(1-5)alkyl, R³ is independently asubstituted or unsubstituted C(6-22)alkyl or C(6-22)alkenyl; R⁴ isindependently a substituted or unsubstituted C(6-22)alkyl orC(6-22)alkenyl; Z is NH, O, or an organic linker consisting of 1-40atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfuratoms; and salts thereof.
 3. The compound of claim 2, wherein Xaa isselected from arginine, homoarginine, norarginine, nor-norarginine,ornithine, lysine, homolysine, histidine, 1-methylhistidine,pyridylalanine, asparagine, N-ethylasparagine, glutamine,4-aminophenylalanine, the N-methylated versions thereof, and side chainmodified derivatives thereof.
 4. The compound of claim 2, wherein Xaa isarginine, an N-methylated version thereof, or a side chain modifiedderivative thereof.
 5. The compound of claim 2, wherein Xaa isnorarginine, an N-methylated version thereof, or a side chain modifiedderivative thereof.
 6. The compound of claim 2, wherein Xaa is lysine,an N-methylated version thereof, or a side chain modified derivativethereof.
 7. The compound of claim 2, wherein Xaa is histidine, anN-methylated version thereof, or a side chain modified derivativethereof.
 8. The compound of claim 2, wherein Xaa is pyridylalanine, anN-methylated version thereof, or a side chain modified derivativethereof.
 9. The compound of claim 2, wherein Xaa is Cysteine or Serine.10. The compound of claim 2, wherein R² is hydrogen.
 11. The compound ofclaim 2, wherein R^(N) is hydrogen.
 12. The compound of claim 2, whereinR³ and R⁴ are C(6-22)alkyl and are the same or different.
 13. Thecompound of claim 2, wherein R³ and R⁴ are C(6-22)alkenyl and are thesame or different.
 14. The compound of claim 2, wherein R³ isC(6-22)alkyl and R⁴ is C(6-22)alkenyl.
 15. The compound of claim 2,wherein R⁴ is C(6-22)alkyl and R³ is C(6-22)alkenyl.
 16. The compound ofclaim 2, wherein Z is NH.
 17. The compound of claim 2, wherein Z is O.18. The compound of claim 2, wherein Xaa is a peptide of 2-20 amino acidresidues.
 19. The compound of claim 2, wherein Xaa has a side chaincontaining a functional group having a pKa from 5 to 7.5.
 20. Thecompound of claim 2, wherein Xaa has a side chain containing a releasingfunctional group selected from 3,5-diiodo-tyrosine, 1-methylhistidine,2-methylbutanoic acid, 2-o-anisylpropanoic acid, meso-tartaric acid,4,6-dimethylpyrimidinamine, p-phthalic acid, creatinine, butanoic acid,N,N-dimethyl-1-naphthylamine, pentanoic acid, 4-methylpentanoic acid,N-methylaniline, 1,10-phenanthroline, 3-pyridinecarboxylic acid,hexanoic acid, propanoic acid, 4-animobenzoic acid, 2-methylpropanoicacid, heptanoic acid, octanoic acid, cyclohexanecarboxylic acid,quinoline, 3-quinolinamine, 2-aminobenzoic acid, 4-pyridinecarboxylicacid, nonanic acid, melamine, 8-quinolinol, trimethylacetic acid,6-methoxyquinoline, 4-(methylamino)benzoic acid, p-methylaniline,3-(methylamino)benzoic acid, malic acid, N-ethylaniline,2-benzylpyridine, 3,6-dinitrophenol, N,N-dimethylaniline,2,5-dimethylpiperazine, p-phenetidine, 5-methylquinoline,2-phenylbenzimidazole, pyridine, picolinic acid, 3,5-diiodityrosine,p-anisidine, 2-(methylamino)benzoic acid, 2-thiazolamine, glutaric acid,adipic acid, isoquinoline, itaconic acid, o-phthalic acid,benzimidazole, piperazine, heptanedioic acid, acridine, phenanthridine,succinic acid, methylsuccinic acid, 4-methylquinoline, 3-methylpyridine,7-isoquinolinol, malonic acid, methymalonic acid, 2-methylquinoline,2-ethylpyridine, 2-methylpyridine, 4-methylpyridine, histamine,histidine, maleic acid, cis-1,2-cyclohexanediamine,3,5-dimethylpyridine, 2-ethylbenzimidazole, 2-methylbenzimidazole,cacodylic acid, perimidine, citric acid, isocitric acid,2,5-dimethylpyridine, papaverine, 6-hydroxy-4-methylpteridine,L-thyroxine, 3,4-dimethylpyridine, methoxypyridine,trans-1,2-cyclohexanediamine, 2,5-pyridinediamine, l-1-methylhistidine,l-3-methylhistidine, 2,3-dimethylpyridine, xanthopterin,1,2-propanediamine, N,N-diethylaniline, alloxanic acid,2,6-dimethylpyridine, L-carnosine, 2-pyridinamine, N-b-alanylhistidine,pilocarpine, 1-methylimidazol, 1H-imidazole, 2,4-dimethylpyridine,4-nitrophenol, 2-nitrophenol, tyrosineamide, 5-hydoxxyquinazoline,1,1-cyclopropanedicarboxylic acid, 2,4,6-trimethylpyridine, veronal,2,3-dichlorophenol, 1,2-ethanediamine, 1-isoquinolinamine, andcombinations thereof.